U.S. patent application number 17/288258 was filed with the patent office on 2021-12-09 for methods and materials for treating neuropsychiatric disorders.
The applicant listed for this patent is Mayo Foundation for Medical Education and Research, University of Newcastle Upon Tyne. Invention is credited to Diana Jurk, James L. Kirkland, Mikolaj B. Ogrodnik, Tamar Tchkonia, Thomas von Zglinicki.
Application Number | 20210379068 17/288258 |
Document ID | / |
Family ID | 1000005836036 |
Filed Date | 2021-12-09 |
United States Patent
Application |
20210379068 |
Kind Code |
A1 |
Kirkland; James L. ; et
al. |
December 9, 2021 |
METHODS AND MATERIALS FOR TREATING NEUROPSYCHIATRIC DISORDERS
Abstract
This document provides methods and materials for treating
obesity-induced neuropsychiatric disorders. For example, one or
more senotherapeutic agents can be administered to a mammal having,
or at risk of developing, an obesity-induced neuropsychiatric
disorder (e.g., obesity-induced anxiety) to treat the mammal.
Inventors: |
Kirkland; James L.;
(Rochester, MN) ; Ogrodnik; Mikolaj B.;
(Rochester, MN) ; Tchkonia; Tamar; (Rochester,
MN) ; Jurk; Diana; (Rochester, MN) ;
Zglinicki; Thomas von; (Whitley Bay, GB) |
|
Applicant: |
Name |
City |
State |
Country |
Type |
Mayo Foundation for Medical Education and Research
University of Newcastle Upon Tyne |
Rochester
Newcastle upon Tyne |
MN |
US
GB |
|
|
Family ID: |
1000005836036 |
Appl. No.: |
17/288258 |
Filed: |
December 18, 2019 |
PCT Filed: |
December 18, 2019 |
PCT NO: |
PCT/US2019/067147 |
371 Date: |
April 23, 2021 |
Related U.S. Patent Documents
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Application
Number |
Filing Date |
Patent Number |
|
|
62782995 |
Dec 20, 2018 |
|
|
|
Current U.S.
Class: |
1/1 |
Current CPC
Class: |
A61P 25/28 20180101;
A61K 31/352 20130101; A61K 31/506 20130101 |
International
Class: |
A61K 31/506 20060101
A61K031/506; A61K 31/352 20060101 A61K031/352; A61P 25/28 20060101
A61P025/28 |
Claims
1. A method for treating an obesity-induced neuropsychiatric
disorder, wherein said method comprises administering a composition
comprising a senolytic agent to a mammal identified as having an
obesity-induced neuropsychiatric disorder.
2. The method of claim 1, wherein said mammal is a human.
3. The method of claim 1, wherein said senolytic agent is dasatinib
or quercetin.
4. The method of claim 1, wherein said composition comprises
dasatinib and quercetin.
5. The method of claim 1, wherein said obesity-induced
neuropsychiatric disorder is obesity-induced anxiety.
6. The method of claim 1, wherein said obesity-induced
neuropsychiatric disorder is obesity-induced depression.
7. The method of claim 1, wherein said composition is effective to
clear senescent cells from within the brain of the mammal.
8. The method of claim 7, wherein said senescent cells comprise an
accumulation of lipids in senescence phenotype.
9. The method of claim 7, wherein said senescent cells are cleared
from in proximity to the lateral ventricle of the brain of the
mammal.
10. The method of claim 1, wherein said composition is effective to
decrease a level of one or more senescence-associated secretory
phenotype (SASP) factor polypeptides in the mammal.
11. A method for increasing neurogenesis, wherein said method
comprises administering a composition comprising a senolytic agent
to a mammal identified as having an obesity-induced
neuropsychiatric disorder under conditions wherein neurogenesis
within said mammal is increased.
12. The method of claim 11, wherein said mammal is a human.
13. The method of claim 11, wherein said senolytic agent is
dasatinib or quercetin.
14. The method of claim 11, wherein said composition comprises
dasatinib and quercetin.
15. The method of claim 11, wherein said obesity-induced
neuropsychiatric disorder is obesity-induced anxiety.
16. The method of claim 11, wherein said obesity-induced
neuropsychiatric disorder is obesity-induced depression.
17. The method of claim 11, wherein said neurogenesis is increased
in the brain of the mammal.
18. The method of claim 17, wherein said neurogenesis is increased
in the subventricular zone of the brain of the mammal.
19. The method of claim 17, wherein said neurogenesis is increased
in the olfactory bulbs of the mammal.
20. The method of claim 11, wherein said composition is effective
to decrease a level of one or more senescence-associated secretory
phenotype (SASP) factor polypeptides in the mammal.
Description
CROSS-REFERENCE TO RELATED APPLICATIONS
[0001] This application claims priority to U.S. Application Ser.
No. 62/782,995, filed on Dec. 20, 2018. The disclosure of the prior
application is considered part of the disclosure of this
application, and is incorporated in its entirety into this
application.
BACKGROUND
1. Technical Field
[0002] This document relates to methods and materials for treating
obesity-induced neuropsychiatric disorders. For example, one or
more senotherapeutic agents can be administered to a mammal having,
or at risk of developing, an obesity-induced neuropsychiatric
disorder (e.g., obesity-induced anxiety) to treat the mammal.
2. Background Information
[0003] Obesity can be associated with a range of neurodegenerative
and psychiatric disorders, including anxiety and depression in some
cases (Gariepy et al., Int J Obes (Lond), 34:407-419 (2010);
Hryhorczuk et al., Front Neurosci, 7:177 (2013); Stunkard and
Wadden, Am J Clin Nutr, 55:524S-532S (1992)). Anxiety is a
behavioral trait in some obese patients (Gariepy et al., Int J Obes
(Lond), 34:407-419 (2010)), affecting 40% more obese patients and
non-obese patients. Increased anxiety-like behavior also was
reported in rodents genetically predisposed to develop obesity,
e.g., db/db mice (Dinel et al., PLoS One 6:e24325 (2011)) and in
high fat (HF) diet-induced obesity (Heyward et al., Neurobiol Learn
Mem 98:25-32 (2012); and Mizunoya et al., Springerplus 2:165
(2013)). Processes such as inflammation (Capuron and Miller,
Pharmacol Ther 130:226-238 (2011); and Lasselin and Capuron, 2014),
altered hormone signaling (Ulrich-Lai and Ryan, Cell Metab
19:910-925 (2014)), and stem cell dysfunction (Anacker and Hen, Nat
Rev Neurosci 18:335-346 (2017); and Gao et al., Neurochem Int
106:24-36 (2017)) have been speculated to underlie obesity-related
anxiety, but the underlying mechanisms have not been
identified.
SUMMARY
[0004] This document provides methods and materials related to
treating obesity-induced neuropsychiatric disorders. For example,
this document provides methods and materials for using one or more
senotherapeutic agents to treat a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder (e.g.,
obesity-induced anxiety). In some cases, a mammal having, or at
risk of developing, obesity-induced anxiety can be treated with a
composition including one or more senotherapeutic agents (e.g.,
dasatinib and/or quercetin) to reduce or eliminate one or more
symptoms of obesity-induced anxiety (e.g., anxiety-like behavior).
In some cases, a mammal having, or at risk of developing,
obesity-induced anxiety can be treated with a composition including
one or more senotherapeutic agents to restore neurogenesis within
the mammal.
[0005] As demonstrated herein, obesity can result in the
accumulation of senescent glial cells in proximity to the lateral
ventricle (LV), a region in which adult neurogenesis occurs, and
these senescent glial cells can exhibit an accumulation of lipids
in senescence (ALISE; e.g., excessive fat accumulation). Also as
demonstrated herein, reducing the level of cells with an ALISE
phenotype from obese mammals (e.g., high fat-fed and leptin
receptor-deficient (db/db) obese mice) can restore neurogenesis and
alleviated anxiety-related behavior. The ability to decrease the
number of senescent glial cells in the LV of an obese mammal (e.g.,
by administering one or more senotherapeutic agents to the mammal)
can be used to treat the mammal having an obesity-induced
neuropsychiatric disorder such as anxiety and depression.
[0006] In general, one aspect of this document features methods for
treating an obesity-induced neuropsychiatric disorder. The methods
can include, or consist essentially of, administering a composition
including a senolytic agent to a mammal identified as having an
obesity-induced neuropsychiatric disorder. The mammal can be a
human. The obesity-induced neuropsychiatric disorder can be
obesity-induced anxiety. The obesity-induced neuropsychiatric
disorder can be obesity-induced depression. The composition can be
effective to clear senescent cells from within the brain of the
mammal. The senescent cells can include an ALISE phenotype. The
senescent cells can be cleared from in proximity to the lateral
ventricle of the brain of the mammal. The composition can be
effective to decrease a level of one or more senescence-associated
secretory phenotype (SASP) factor polypeptides in the mammal.
[0007] In another aspect, this document features methods for
increasing neurogenesis. The methods can include, or consist
essentially of, administering a composition including a senolytic
agent to a mammal identified as having an obesity-induced
neuropsychiatric disorder under conditions wherein neurogenesis
within the mammal is increased. The mammal can be a human. The
obesity-induced neuropsychiatric disorder can be obesity-induced
anxiety. The obesity-induced neuropsychiatric disorder can be
obesity-induced depression. The neurogenesis can be increased in
the brain of the mammal. For example, the neurogenesis can be
increased in the subventricular zone of the brain of the mammal.
For example, the neurogenesis can be increased in the olfactory
bulbs of the mammal. The composition can be effective to decrease a
level of one or more SASP factor polypeptides in the mammal.
[0008] Unless otherwise defined, all technical and scientific terms
used herein have the same meaning as commonly understood by one of
ordinary skill in the art to which this invention pertains.
Although methods and materials similar or equivalent to those
described herein can be used to practice the invention, suitable
methods and materials are described below. All publications, patent
applications, patents, and other references mentioned herein are
incorporated by reference in their entirety. In case of conflict,
the present specification, including definitions, will control. In
addition, the materials, methods, and examples are illustrative
only and not intended to be limiting.
[0009] The details of one or more embodiments of the invention are
set forth in the accompanying drawings and the description below.
Other features, objects, and advantages of the invention will be
apparent from the description and drawings, and from the
claims.
DESCRIPTION OF THE DRAWINGS
[0010] FIG. 1 shows that obese mice exhibit anxiety-like behavior
that is not directly related to an increase in body mass.
Behavioral changes were tested in the Open Field (OF) chamber. Dark
rectangle marks the central area (25% of total area). (A)
Representative movement traces (red lines) for chow- and HF diet
(HFD)-fed mice at 10 months of age (baseline). Parameters recorded
and analyzed in OF: (B) distance travelled in the central area (as
a function of total distance travelled) and (C) entries into the
central area. No significant correlations (linear regression) were
found in both chow or HFD animals between (D) body mass and the
normalized distance mice travelled in the central area and (E) the
number of entries into the central area. (F) Representative heat
maps of time mice spent in the elevated plus maze (EPM) for chow
and HFD mice. Closed arms of the maze are indicated by pink
brackets. (G) The frequency of head pokes into open arms and (H)
the time mice spent with their head in open arms are significantly
decreased in HFD- when compared to chow-fed mice. No correlations
(linear regression) were found between body mass and elevated plus
maze test parameters in chow and HFD mice: (I) frequency of head
pokes into open arms and (J) time spent in open arms. Data are from
n=26-30 mice per group, Mean.+-.SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
[0011] FIG. 2 shows that linear regression analysis revealed no
significant correlations between anxiety-like behavior markers,
body weight, and % of fat in obese mice. (A) Body mass and (B)
normalized body fat in chow- and HFD mice. (C) The total distance
travelled and (D) total time spent in the central zone of open
field in chow- and HFD mice. Linear regression analysis between
body mass (E), total distance travelled in open field, and (F)
total time spent in the central zone of the open field in lean and
obese mice. Linear regression analysis between % of body fat and
anxiety-like behavior parameters in the open field test: (G)
normalized distance travelled in the central area, (H) entries to
the central zone, (I) total distance travelled during the test, and
(J) time spent in the central area. Body fat of chow- and HFD mice
was correlated to parameters of anxiety-like behavior measured in
the elevated plus maze test: (K) frequency of head pokes into the
open arms and (L) time of head spend in open arms. Data are from:
n=26-30 mice per group, Mean.+-.SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
[0012] FIG. 3 shows pharmacogenetic and pharmacologic clearance of
senescent cells from obese mice alleviates obesity-related
behavioral changes. (A) Eight month-old C57Bl/6.sup.(INK-ATTAC)
male mice were split into four groups and assigned to chow (n=24)
or high fat (HF, n=30) diet and treated at 10 months of age with
vehicle (n=12 for chow and n=15 for HF) or AP20187 (n=12 for chow
and n=15 for HF) until 13 months of age. (B) Open field testing:
Representative movement traces (red lines) of lean and obese
INK-ATTAC mice treated with/without AP20187 (AP) indicates that
anxiety-like behavior of HFD mice can be alleviated by AP treatment
as measured by (C) normalized distance travelled in the central
area of the open field box and (D) increased frequency of entries
into the central area. Alleviation of anxiety-like behavior of HFD
mice was also observed by elevated plus maze test. (E)
Representative heat-map images of time mice spent in open and
closed sections of the elevated plus maze. Parameters were
registered by EthoVision software. (F) Frequency of head pokes into
the open arms and (G) time mice spent with their heads in open
area, was significantly reduced with HFD and increased with AP. (H)
Representative movement traces (red lines) of db/db and
heterozygous control mice with or without treatment with senolytic
cocktail dasatinib+quercetin (D+Q) in the open field test during a
30 minute trial. Data indicates an improvement of anxiety-like
behavior of db/db mice upon D+Q treatment determined by open field
test parameters: (I) normalized distance travelled in the middle
area and (J) frequency of entries into the middle area of the open
field box. In the open field test, INK-ATTAC;db/db mice (K)
displayed a significant difference in the distance travelled in the
central area after treatment with AP20187 when compared to vehicle,
but (L) the number of entries into the central area was not
significantly increased after treatment with AP. Data are from
n=12-15 mice per group for A-G; n=8-12 mice per group for H-J;
n=5-9 mice per group for K-L. Mean.+-.SEM plotted. * P.ltoreq.0.05
and ** P.ltoreq.0.001.
[0013] FIG. 4 shows additional phenotypic and molecular features of
AP20187-treated INK-ATTAC mice. (A) Body mass measurements of mice
on chow and HF diet before and after last treatment with AP20187
(AP) shows no change in body weight over time. Additional
parameters from the open field test: (B) distance in the middle
area as a function of the total distance travelled and to the
baseline (measurements before the treatment) and (C) total distance
travelled. Normalized to baseline parameters of elevated plus maze
testing: (D) head pokes toward the open arms and (E) time spent
with the head in the open area of the maze. (F, G) Short-term
memory was not affected by obesity or AP treatment as determined by
lack of substantial changes in Stone's maze parameters: (F) time
needed to finish the maze and (G) frequency of errors. C57Bl6
wild-type mice were used to test off-target effects of AP. (H)
Before treatment mice under HF diet showed anxiety-like behavior
(measured by distance travelled in the center of the open field
box). (I) Treatment of C57Bl6 mice under HF diet with AP showed no
difference in behavior between treated and un-treated mice. Db/db
and heterozygous lean (db/+) mice were given 2 months of D+Q
treatment for 5 days every 2 weeks starting from the age of 4
months. Db/db mice did not show changes in (J) body mass or (K)
body fat within the groups over the course of treatment. (L) Total
distance travelled by db/db and db/+ mice in the open field test
was not affected by D+Q treatment. 3-month old INK-ATTAC;
INK-ATTAC:db/db mice were randomly sorted to AP or vehicle groups
and treated for 2 months. No changes in (M) body mass or (N) body
composition were observed in AP treated mice. (0) Total distance
travelled during 30 minute long open field testing was not affected
by AP treatment. (P) Db/db mice were tested for off-target effects
of AP. Treatment showed no difference in behavior between treated
and un-treated mice. (Q-S) Linear regression analysis between body
weight and anxiety-like behavior, measured by distance travelled in
the centre of the open field box, showed no association between the
two in HF diet INK-ATTAC mice treated with and without AP (Q), in
db/db mice treated with and without D+Q and (S) in INK-ATTAC:db/db
mice treated with and without AP20187. Data are from n=13-15 mice
per group for graphs A-E, n=9-24 mice per group for F-G, n=7-12
mice per group for H, n=9-10 mice per group for graphs I, n=6-8
mice per group for graphs J-L, n=8-9 mice per group for graphs M-N,
n=16 mice per group for the graph O, n=11 mice per group for the
graph P, n=30-31 mice per group for graphs Q and S and n=23 mice
per group for the graph R. Mean.+-.SEM plotted. * P.ltoreq.0.05 and
** P.ltoreq.0.001.
[0014] FIG. 5 shows that decreasing the amount of senescent cells
in obese animals reduces circulating cytokine levels. (A)
Quantification of % of senescence-associated beta-galactosidase
(SA-.beta.-Gal) positive cells in perigonadal adipose tissue shows
increased values in HF diet-fed animals and complete rescue after
treatment with AP20187. Senescent markers p16 (measured by RT-PCR)
(B) and telomere associated DNA damage foci (TAF) (C) show a
similar pattern. (D) Representative images and (E) quantification
of SA-.beta.-Gal activity in perigonadal adipose tissue of db/db
and db/+ mice shows that % of SA-.beta.-Gal positive cells increase
in db/db mice compared to db/+ and are significantly reduced upon
treatment with the senolytic cocktail D+Q. (F) Frequencies of
TAF-positive cells in perigonadal adipose tissue of db/db increase
significantly in comparison to db/+ mice and decrease after D+Q
treatment. (G) Cytokine protein expression [fold change] in blood
plasma from HFD animals treated with/without AP20187. (H) Cytokine
expression [fold change] in blood plasma from db/db animals treated
with/without AP20187. Linear regression analysis between anxiety
markers and cytokines in blood plasma showed a significant negative
correlation between (I) Cxcl-1, (J) G-Csf, (K) Mig and the distance
travelled in the central zone of the open field box in HF diet fed
mice. Data are from n=6-9 mice per group for A-C and G-H; n=7-12
mice per group for D-F and I-J; n=27 mice per group for K-M.
Mean.+-.SEM plotted. * P.ltoreq.0.05 and ** P.ltoreq.0.001.
[0015] FIG. 6 shows influence of systemic factors on anxiety-like
behavior. Quantification of p21 (A) by PCR and the number of DNA
damage foci (.gamma.-H2A.X) (B) by IF-staining in perigonadal
adipose tissue shows increased values in HF-diet fed animals and
but no change after treatment with AP20187. Correlations between
anxiety-like phenotype markers and Cxcl-1 in blood plasma of HFD
and chow animals (C) in EPM, (D) OF and in db/db and db/db.sup.+/-
animals (E) EPM. Correlations between (F) Tnf-.alpha., (G) Il-6 and
(H) Mcp-1 in blood plasma of HF and chow diet animals and the
distance travelled in the central zone of the open field box showed
no significant differences. To test the effect of increased or
decreased cytokine levels in the blood stream mice were injected
with Cxcl-1 or treated with Cxcr1 inhibitor Reparixin (I) Cxcl-1 is
significantly increased in blood plasma of C57Bl6 mice injected
with Cxcl-1 in comparison to vehicle-treated mice. Mice injected
with Cxcl- lost weight (J) but did not experience changes in body
fat (K). Distance travelled in the central area of the open field
box (L) and head entries in the open arms of the EPM (M) were not
different between Cxcl-1 and vehicle treated mice. (N) Mice under
high fat diet and treated with Reparixin or vehicle showed an
increase in body mass, but did show no alterations in body fat (0).
No difference in the open field (P) and EPM (Q) test were observed.
(R) Scheme showing experimental setup for transplantation
experiments. C57Bl6 mice were divided into 3 groups, injected
either with PBS, young or senescent mouse preadipocytes and tested
for anxiety-like behavior (OF) and frailty (Rotarod). (S) Rotarod
testing showed significant decreased performance in mice injected
with senescent cells at 12 weeks but not 2 weeks after injection.
All mice showed no anxiety-like phenotype 2 and 6 weeks after cell
transplantation when distance travelled (T), entries (U),
normalized distance travelled in the central area and total
distance travelled (V, W) in the open field box were measured. Data
are from n=6-8 mice per group for graphs A-B, mice per group n=27
for graphs C and F-H, mice per group n=36 for graphs D-E, n=10 mice
per group for graphs I-M, n=12 mice per group for graphs N-Q and
n=6-7 mice per group for the graphs S-W. Mean.+-.SEM plotted. *
P.ltoreq.0.05 and ** P.ltoreq.0.001.
[0016] FIG. 7 shows markers of senescence in the amygdala are
reduced after treatment with AP20187. (A) Cdkn2a positive cells
were measured by RNA-ISH in the basomedial layer of the amygdala.
(B) Representative images showing telomere associated DNA damage
foci (TAF), (blue=DAPI, red=telomeres, green=.gamma.-H2A.X, white
arrow indicates TAF) in 3 .mu.m thick paraffin embedded brain
sections. (C) Mean number of TAF and (D) % of NeuN-pos cells with 2
or more TAF was increased in HFD INK-ATTAC mice and significantly
reduced after AP20187 treatment in the basomedial layer of the
amygdala. (E) Mean number of TAF and (F) % of NeuN-neg cells with 2
or more TAF was increased in HFD INK-ATTAC mice and significantly
reduced after AP20187 treatment in the hypothalamus in close
proximity to the 3.sup.rd ventricle. Data are from n=5-6 mice per
group for graphs B-D; Mean.+-.SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
[0017] FIG. 8 shows levels of senescent markers are not changed in
cortex, cerebellum, or hippocampus of HFD when compared to lean
animals. Analysis of p21 (A) and p16 (B) by RT-PCR in different
brain regions (cortex, cerebellum, and hippocampus) revealed no
significant difference among groups. Quantification of
.gamma.-H2A.X foci (C) and telomere associated damage foci (TAF)
(D) in neurons in different brain areas showed no significant
difference. (E) Mean number of TAF and (F) % of NeuN-pos cells with
2 or more TAF was increased in HFD INK-ATTAC mice and significantly
reduced after AP20187 treatment in the hypothalamus in close
proximity to the 3.sup.rd ventricle. Data are from n=5-8 mice per
group for graphs A-D; n=5 for E, F. Mean.+-.SEM plotted. *
P.ltoreq.0.05 and ** P.ltoreq.0.001.
[0018] FIG. 9 shows obesity-related accumulation of lipid droplets
in senescent periventricular glia is reduced upon senescent cells
clearance. (A) Representative images of Perilipin 2 (Plin2)
staining showing accumulation of cells exhibiting build-up of lipid
droplets in close proximity to the lateral ventricle (LV) of
middle-aged, obese mice compared to their lean littermates. (B)
Quantification of frequencies of Plin2.sup.+ cells in the proximity
(up to 250 .mu.m from ependymal cell layer) to the LV in high fat
(HF)-fed and lean mice. (C) Representative images showing
Plin2.sup.+ cells co-localizing with markers of microglia (Iba1;
top left panel) and astrocytes (vimentin [Vim]; top right panel)
but not with neuronal markers (NeuN; bottom panel). (D) Pie chart
shows cell-type composition of Plin2.sup.+ cells [determined after
Immunostaining for Plin2 and different cell-type markers (as shown
in C)]. (E) Periventricular Plin2.sup.+ glial cells show increased
numbers of senescent-marker telomere associated foci (TAF).
Quantification of mean number of TAF per cell in non-neuronal
(NeuN.sub.neg) Plin.sup.+ and Plin.sup.- cells. (F) Representative
Images show the LV of chow (top panel) and HF AP20187-treated mice
stained with Plin2, exhibiting reduced lipid droplets. (G)
Quantification of cells containing lipid droplets (Plin2.sup.+) in
the periventricular area of lean HF INK-ATTAC mice with or without
AP20187 treatment. (H) Quantification of frequencies of NeuN
negative, TAF-positive cells in the periventricular region of
lean/HF and vehicle or AP20187-treated mice. (I) Representative
images showing double staining for CXCL1 (RNA-ISH in red) and Plin2
(green) in periventricular area of HF INK-ATTAC mice. White arrows
indicate CXCL1 and Plin2 double positive cells. Cells magnified in
panel 4 encircled in red show positive staining for Plin2 and
CXCL1, whereas white-bordered cells are not double positive.
Quantification of cells positive for Cxcl1 (J) and Il-6 (K) stained
by RNA-ISH indicate that Plin.sup.+ cells display significantly
higher expression levels of senescence-associated secretory
phenotype (SASP) factors Il-6 and Cxcl1 than Plin.sup.- cells.
Build-up of fat in periventricular brain region as assessed by
Plin2 staining significantly correlates with parameters associated
with anxiety-like behavior: (L) % of distance travelled in the
central zone (% of central zone) and (M) number of entries into the
central zone (entries) in open field testing. Data are from n=5-8
mice per group for B, n=6 mice per group for E, J and K, n=4-8 mice
per group for G-H, and n=25 mice per group for L and M. Mean.+-.SEM
plotted. *P.ltoreq.0.05 and ** P.ltoreq.0.001.
[0019] FIG. 10 shows assessment of periventricular fat accumulation
and markers of senescence in lean and obese mice. (A) Lipid
droplets in periventricular cells visualized by Perilipin 2 (Plin2)
staining. The panel on the top right shows a magnified cell and the
panel on the bottom right shows magnified lipid droplets visualized
by Plin2 staining. (B) Plin2 staining shows accumulation of lipid
droplets in cells in close proximity to the 3.sup.rd (left panel),
the 4.sup.th ventricle (middle panel) and periaqueductal grey
matter (PAG) (right panel). Bottom left images show merged images
of Plin2 and DAPI staining. (C) .gamma.-H2A.X foci were quantified
in Plin2.sup.+ and Plin2.sup.-, non-neuronal cells in the lateral
ventricle. (D) Images showing the ependymal layer in the LV of
db/db vehicle (top panel) and db/db D+Q-treated mice stained with
Plin2 in red and S100.beta. in green. Areas in white rectangles are
magnified on the right and show reduction in size of Plin2 lipid
droplets in D+Q treated animals. (E) Quantification of the area
containing lipid droplets (Plin2.sup.+) in the ependymal layer of
db/db and db/db.sup.-/- mice with or without D+Q treatment. (F)
Quantification of frequencies TAF-positive cells in the ependymal
layer of db/db and db/+ and vehicle or D+Q-treated mice. Data are
from n=6 mice per group for graph C, n=7-12 mice per group for
graph E, F. Mean.+-.SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
[0020] FIG. 11 shows ALISE phenotype drives CCF accumulation and
SASP. Similarly to glia in the brains of obese mice, mouse adult
fibroblasts (MAFs) show an Accumulation of Lipids In SEnescence
(ALISE) phenotype. (A-F) Depletion of lipids from culture media
reduces area of lipid droplets in MAFs. (A) ALISE phenotype in MAFs
is characterised by increased area of lipid droplets surrounded by
Plin2 vesicles. (B) Quantification of Nile Red-positive staining in
young (you) and senescent (sen) MAFs. (C-D) Suppression of ALISE
phenotype reduces frequencies of Cytoplasmic Chromatin Fragments
(CCF) in senescent fibroblasts but (E-F) does not affect number of
53BP1 DNA damage foci. (G-K) Senescent fibroblasts have increased
secretion of SASP components including Il-6, KC (Cxcl1), and Ip-10
(Cxcl10) that is alleviated upon suppression of ALISE phenotype.
(G) Heat map shows fold change in secretion of SASP components to
cell culture media over a 72 hour time-period when compared to
young fibroblasts cultured with lipid-containing media. Each square
represents a separate biological replicate, MAFs isolated from a
different mouse donor. Concentration of SASP components in culture
media with and without lipids (ALISE phenotype suppression) for (H)
Il-6, (I) Ip-10 (Cxcl10), (J) Vegf, and (K) Kc (Cxcl1). (L) Images
show accumulation of lipid droplets in senescent but not young
GFAP-positive astrocytes. (M-O) Characterization of senescence in
astrocytes. Senescent astrocytes show (M) the ALISE phenotype
measured using Nile Red staining, (N) increased frequencies of
telomere dysfunction measured as frequency of cells with 1 or more
telomere associated DNA damage foci (TAF), and (O) increased
frequencies of CCF. Senescence was induced by X-ray irradiation (10
Gy) and established within 14-21 days post-irradiation. Data are
from n=3-6 mice per group. Mean.+-.SEM plotted. * P.ltoreq.0.05 and
** P.ltoreq.0.001.
[0021] FIG. 12 shows an analysis of ALISE phenotype in astrocytes.
(A) Suppression of the ALISE phenotype by culturing cells in lipid
free media doesn't change bi-nuclearity in MAFs. In astrocytes, the
average number of TAF (B), average number of 53BP1 foci (C) and
bi-nuclearity (D) increases significantly after induction of
senescence. Senescence was induced by X-ray irradiation (10 Gy) and
established within 14-21 days post-irradiation. Data are from n=3
mice. Mean.+-.SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
[0022] FIG. 13 shows clearance of senescent cells partially
reverses the neural progenitor cell depletion induced by obesity.
(A) Each hemisphere of INK-ATTAC lean and high fat (HF) mouse brain
was either dissociated into a single-cell suspension or processed
for IHC/IF. Dissociated brain cells were labelled with
metal-conjugated antibodies and processed for Cytometry by Time of
Flight (CyTOF). (B, D) Spanning-tree Progression Analysis of
Density-normalized Events (SPADE) was performed on brain cell
populations identified by markers shown on the micrographs. Heat
map shows the intensity of antibody signal and the size of each
spot is determined by the number of cells within this population.
(C) CyTOF shows differences in brain cell populations of INK-ATTAC
chow and HF mice, which were treated with vehicle or AP20187.
Frequencies of cells expressing markers (E) doublecortin (Dcx), (F)
CD133 and (G) Nestin were quantified. (H) Representative images of
doublecortin (Dcx) staining in the olfactory bulb of chow- and HFD
mice treated with vehicle or AP20187. White boxes show magnified
regions. (I) Quantitative analysis of Dcx-positive area of the
granular layer in the olfactory lobe of lean and obese INK-ATTAC
mice and (J) correlation between area occupied by Dcx.sup.+ cells
in the granular layer of the olfactory bulb and frequencies of
Dcx.sup.+ cells from the whole brain measured by CyTOF.
Correlations (linear regression analysis) between frequencies of
periventricular glia exhibiting accumulation of lipid droplets and
frequencies of cells expressing markers of neurogenesis and
ependymal cells (determined by CyTOF): (K) Nestin and (L) Dcx.
Correlations (linear regression analysis) between distance
travelled in the central zone and frequencies of cells expressing
(M) Nestin and (N) Dcx (determined by CyTOF). Data are from n=5-9
mice per group for C-G; n=2-6 mice per group for I; n=11 mice per
group for J; n=29 mice per group for K-N. Mean.+-.SEM plotted. *
P.ltoreq.0.05 and ** P.ltoreq.0.001.
[0023] FIG. 14 shows association between adult neurogenesis and
periventricular lipid accumulation. (A) Cells displaying perilipin
2 (Plin2)-positive lipid droplets were found in close proximity to
doublecortin (Dcx)-positive cells. Yellow box marks magnified
region shown on the right. Characterization by Cytometry by Time of
Flight (CyTOF) of different cell-types in the brain of lean and
obese INK-ATTAC mice with or without AP treatment. Quantification
of markers of oligodentrocytes: (B) 2',3'-cyclic-nucleotide
3'-phosphodiesterase (CNPase), (C) oligodendrocyte specific protein
(OSP), (D) double positive cells for CNPase and OSP; markers of
microglia: (E) CD11b and (F) CD45.sup.-/CD11b.sup.+; markers of
astrocytes: (G) astrocyte cell surface antigen-2 (ACSA-2), (H)
glial fibrillary acidic protein (Gfap), and (I) double-positive for
ACSA-2 and Gfap; (J) marker of astrocytes, epithelial cells,
pericytes, and ependymal cells vimentin (Vim); markers of
endothelial cells and pericytes: (K) CD146 and (L) CD31; and a (M)
marker of neurons, NeuN. One hemisphere of each db/db vehicle or
D+Q treated mouse brain was dissociated into a single-cell
suspension and labelled with metal-conjugated antibodies and
processed for (CyTOF). Frequencies of cells expressing markers (N)
doublecortin (Dcx), (0) CD133 and (P) Nestin were quantified. (Q)
GFAP staining (blue is DAPI) in the cerebral cortex and (R) its
quantification (in the indicated areas). (S) Representative images
of EdU staining in the preventricular area of chow- and HF diet-fed
mice treated with vehicle or AP20187. Yellow boxes show magnified
regions. (T) Quantitative analysis of EdU-positive cells per image
in lean and obese INK-ATTAC mice with or without AP treatment. (U)
A representative image of immunofluorescent staining for DCX in the
dentate gyrus (DG) of the hippocampus. (V) Frequencies of
DCX-positive cells in the DG and (W) total amount of EdU-positive
cells in the hippocampus per hemisphere in lean and HFD INK-ATTAC
animals with and without AP treatment. Data are from n=6-9 mice per
group for graphs B-M, n=8 mice per group for graphs N-P, n=5-8 mice
per group for the graph R, n=3 mice per group for the graph T,
n=5-8 mice per group for the graph V, n=6 mice per group for the
graph W. Mean+/-SEM plotted. * P.ltoreq.0.05 and **
P.ltoreq.0.001.
DETAILED DESCRIPTION
[0024] This document provides methods and materials related to
treating obesity-induced neuropsychiatric disorders. For example,
this document provides methods and materials for using one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) to treat
a mammal having an obesity-induced neuropsychiatric disorder (e.g.,
obesity-induced anxiety). In some cases, one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) can be
used as described herein to treat a mammal at risk of developing an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety).
[0025] In some cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder can be treated with a
composition including one or more senotherapeutic agents (e.g.,
dasatinib and/or quercetin) to alleviate (e.g., to reduce or
eliminate) one or more (e.g., one, two, three, four, five, or more)
symptoms of the obesity-induced neuropsychiatric disorder. An
obesity-induced neuropsychiatric disorder can be any type of
obesity-induced neuropsychiatric disorder. Examples of
obesity-induced neuropsychiatric disorder include, without
limitation, obesity-induced anxiety, obesity-induced depression,
obesity-induced fearfulness, obesity-related suicide, and
obesity-induced stress. A symptom of an obesity-induced
neuropsychiatric disorder can be any appropriate symptom. For
example, examples of symptoms of obesity-induced anxiety include,
without limitation, anxiety-related behaviors such as feeling
nervous, feeling restless, feeling tense, feeling stressed, having
a sense of impending danger, increased heart rate,
hyperventilation, sweating, trembling, feeling weak or tired,
trouble concentrating or thinking about anything other than the
present worry, having trouble sleeping, and gastrointestinal
problems. Each of these symptoms of obesity-induced anxiety can be
identified, staged, and/or monitored using clinical techniques as
described elsewhere (see, e.g., Practice Guidelines for Psychiatric
Evaluation of Adults, Third Edition, American psychiatric
association, 2016; Lykouras et al., Psychiatriki 22:307-13 (2011);
and Locke et al., Am Fam Physician 91:617-24 (2015)). For example,
examples of symptoms of obesity-induced depression include, without
limitation, feelings of sadness, feelings of tearfulness, feelings
of hopelessness, feelings of worthlessness, angry outbursts,
irritability or frustration, loss of interest or pleasure normal
activities hobbies or sports, sleep disturbances, lack of energy,
fixating on past failures or self-blame, frequent or recurrent
thoughts of death, suicidal thoughts, suicide attempts, and
unexplained physical problems such as back pain or headaches. Each
of these symptoms of obesity-induced depression can be identified,
staged, and/or monitored using clinical techniques as described
elsewhere (see, e.g., Practice Guidelines for Psychiatric
Evaluation of Adults, Third Edition, American psychiatric
association, 2016; and Clinical Practice Guidelines, American
Psychiatric Association, available at
psychiatry.org/psychiatrists/practice/clinical-practice-guidelines)
In some cases, administering one or more senotherapeutic agents to
a mammal having, or at risk of developing, an obesity-induced
neuropsychiatric disorder can be effective to alleviate one or more
anxiety-related behaviors in the mammal.
[0026] In some cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) can be treated with a composition including one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) to clear
one or more senescent cells from within the mammal. A senescent
cell can be any type of cell. In some cases, a senescent cell can
exhibit excessive fat accumulation (e.g., can have an ALISE
phenotype). Examples of senescent cells that can be cleared as
described herein include, without limitation, a senescent glial
cell, an ependymal cell, a neural progenitor cell, a neuron, and an
endothelial cell. A senescent cell can be cleared from any location
within the mammal. In some cases, a senescent cell can be cleared
from the brain of a mammal. Examples of locations from which a
senescent cell cleared include, without limitation, in proximity to
the LV of the brain of the mammal, in the LV of the brain of the
mammal, in proximity to the subventricular zone (SVZ) of the brain
of the mammal, in the SVZ of the brain of the mammal, and cerebral
blood vessels. A location in proximity to the LV of the brain of a
human can be the region within about 10 mm (within about 9 mm,
within about 8 mm, within about 7 mm, within about 6 mm, within
about 5 mm, within about 4 mm, within about 3 mm, within about 2
mm, or within about 1 mm) of the LV. A location in proximity to the
SVZ of the brain of a human can be the region within about 10 mm
(within about 9 mm, within about 8 mm, within about 7 mm, within
about 6 mm, within about 5 mm, within about 4 mm, within about 3
mm, within about 2 mm, or within about 1 mm) of the SVZ. In some
cases, administering one or more senotherapeutic agents to a mammal
having, or at risk of developing, an obesity-induced
neuropsychiatric disorder can be effective to clear one or more
senescent cells having an ALISE phenotype from a location in
proximity to the LV of the brain of the mammal.
[0027] In some cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) can be treated with a composition including one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) to
increase (e.g., restore) neurogenesis within the mammal. In some
cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) can be treated with a composition including one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) to
alleviate (e.g., to reduce or eliminate) obesity-related impairment
of neurogenesis in the mammal. Neurogenesis of any appropriate type
of cell can be increased. Examples of cells for which neurogenesis
can be increased as described herein include, without limitation,
neuronal precursor cells, immature neurons, ependymal cells, and
developing neurons. Neurogenesis can be increased in any location
within the mammal. Examples of locations in which a neurogenesis
can be increased include, without limitation, in the SVZ of the
brain of the mammal, and in the olfactory bulbs of the mammal. In
some cases, administering one or more senotherapeutic agents to a
mammal having, or at risk of developing, an obesity-induced
neuropsychiatric disorder can be effective to restore neurogenesis
in the mammal.
[0028] In some cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) can be treated with a composition including one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) to
alleviate (e.g., to reduce or eliminate) inflammation in the
mammal. A level (e.g., a systemic level) of any appropriate
inflammatory factor (e.g., cytokines, chemokines, and matrix
proteases) can be altered (e.g., increased or decreased) to
alleviate inflammation in a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder. In cases
where an inflammatory factor is a pro-inflammatory factor (e.g.,
SASP factor polypeptides such as G-Csf, Il-1.alpha. and Il-1.beta.,
Kc/Cxcl1, Mcp-1, Mig, Il-6, Tnf-.alpha.; and IL-8) the
pro-inflammatory factor can be decreased. In cases where an
inflammatory factor is an anti-inflammatory factor, the
anti-inflammatory factor can be increased. Inflammation at any
appropriate location within the mammal can be alleviated. Examples
of locations from which inflammation can be alleviated as described
herein include, without limitation, the brain, blood vessels,
adipose tissue, the lungs, kidneys, the liver, bone, bone marrow,
and skin. In some cases, a systemic inflammatory factor (e.g.,
systemic SASP factor polypeptides) can cross the blood-brain
barrier to alleviate brain inflammation. In some cases,
administering one or more senotherapeutic agents to a mammal
having, or at risk of developing, an obesity-induced
neuropsychiatric disorder can be effective to alleviate brain
inflammation within the mammal.
[0029] In some cases, when a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder (e.g.,
obesity-induced anxiety) is treated with a composition including
one or more senotherapeutic agents (e.g., dasatinib and/or
quercetin), the mammal's body weight is not affected (e.g., is not
altered).
[0030] In some cases, when a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder (e.g.,
obesity-induced anxiety) is treated with a composition including
one or more senotherapeutic agents (e.g., dasatinib and/or
quercetin), the mammal's body composition is not affected (e.g., is
not altered).
[0031] In some cases, when a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder (e.g.,
obesity-induced anxiety) is treated with a composition including
one or more senotherapeutic agents (e.g., dasatinib and/or
quercetin), the mammal's activity is not affected (e.g., is not
altered).
[0032] When treating a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) as described herein (e.g., by administering one or more
senotherapeutic agents such as dasatinib and/or quercetin), the
mammal can be any appropriate mammal. In some cases, a mammal can
be an obese mammal (e.g., a mammal that is overweight). Examples of
mammals that can be treated using a composition containing one or
more senotherapeutic agents as described herein include, without
limitation, humans, non-human primates such as monkeys, dogs, cats,
horses, cows, pigs, sheep, mice, and rats. In some cases, a
composition containing one or more senotherapeutic agents can be
administered to a human having an obesity-induced neuropsychiatric
disorder to treat the human. In some cases, a composition
containing one or more senotherapeutic agents can be administered
to a human at risk of developing an obesity-induced
neuropsychiatric disorder to slow the onset or progression of an
obesity-induced neuropsychiatric disorder within the human.
[0033] In some cases, the methods described herein also can include
identifying a mammal as having, or as being at risk of developing,
an obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety). Examples of methods for identifying a mammal as having,
or as being at risk of developing, an obesity-induced
neuropsychiatric disorder include, without limitation,
psychological evaluation, physical examination, and/or laboratory
tests such as stress hormone levels. Once identified as having, or
as being at risk of developing, an obesity-induced neuropsychiatric
disorder, a mammal can be administered or instructed to
self-administer one or more senotherapeutic agents (e.g., dasatinib
and/or quercetin).
[0034] A composition containing one or more (e.g., one, two, three,
four, five, or more) senotherapeutic agents can include any
appropriate senotherapeutic agent(s). A senotherapeutic agent can
be any type of molecule (e.g., small molecules or polypeptides). In
some cases, a senotherapeutic agent can be a senolytic agent (i.e.,
an agent having the ability to induce cell death in senescent
cells). In some cases, a senotherapeutic agent can be a senomorphic
agent (i.e., an agent having the ability to suppress senescent
phenotypes without cell killing). Examples of senotherapeutic
agents that can be used as described herein (e.g., to treat a
mammal having, or at risk of developing, an obesity-induced
neuropsychiatric disorder such as obesity-induced anxiety) can
include, without limitation, dasatinib, quercetin, navitoclax,
A1331852, A1155463, fisetin, luteolin, geldanamycin, tanespimycin,
alvespimycin, piperlongumine, panobinostat, FOX04-related peptides,
nutlin3a, ruxolitinib, metformin, and rapamycin.
[0035] In some cases, a composition containing one or more (e.g.,
one, two, three, four, five, or more) senotherapeutic agents (e.g.,
dasatinib and/or quercetin) can include the one or more
senotherapeutic agent(s) as the sole active ingredient(s) in the
composition that is effective to treat an obesity-induced
neuropsychiatric disorder (e.g., obesity-induced anxiety). In some
cases, a composition containing one senotherapeutic agent (e.g.,
fisetin) can include that one senotherapeutic agent as the sole
active ingredient in the composition that is effective to treat an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety).
[0036] In some cases, a composition containing one or more (e.g.,
one, two, three, four, five, or more) senotherapeutic agents (e.g.,
dasatinib and/or quercetin) can include one or more (e.g., one,
two, three, four, five, or more) additional active agents (e.g.,
therapeutic agents) in the composition that are effective to treat
an obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety).
[0037] In some cases, a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety) being treated as described herein (e.g., by administering
one or more senotherapeutic agents such as dasatinib and/or
quercetin) also can be treated with one or more (e.g., one, two,
three, four, five, or more) additional therapeutic agents. A
therapeutic agent used in combination with one or more
senotherapeutic agents described herein can be any appropriate
therapeutic agent. Examples of therapeutic agents that can be used
in combination with one or more senotherapeutic agents described
herein include, without limitation, benzodiazepines (e.g.,
alprazolams such as XANAX.TM., chlordiazepoxides such as
LIBRIUIM.RTM., clonazepams such as KLONOPIN.RTM., diazepams such as
VALIUM.RTM., and lorazepams such as ATIVAN.RTM.), buspirone, and
antidepressants including selective serotonin reuptake inhibitors
(SSRIs; e.g., escitaloprams such as LEXAPRO, fluoxetines such as
PROZAC.RTM., paroxetines such as PAXIL.RTM., and sertralines such
as ZOLOFT.RTM.). In some cases, the one or more additional
therapeutic agents can be administered together with the one or
more senotherapeutic agents (e.g., in a composition containing one
or more senotherapeutic agents and containing one or more
additional therapeutic agents). In some cases, the one or more
(e.g., one, two, three, four, five, or more) additional therapeutic
agents can be administered independent of the one or more
senotherapeutic agents. When the one or more additional therapeutic
agents are administered independent of the one or more
senotherapeutic agents, the one or more senotherapeutic agents can
be administered first, and the one or more additional therapeutic
agents administered second, or vice versa.
[0038] In some cases, a composition containing one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) can be
formulated into a pharmaceutically acceptable composition for
administration to a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety). For example, one or more senotherapeutic agents can be
formulated together with one or more pharmaceutically acceptable
carriers (additives) and/or diluents. Pharmaceutically acceptable
carriers, fillers, and vehicles that can be used in a
pharmaceutical composition described herein include, without
limitation, saline, ion exchangers, alumina, aluminum stearate,
lecithin, serum proteins, such as human serum albumin, buffer
substances such as phosphates, glycine, sorbic acid, potassium
sorbate, partial glyceride mixtures of saturated vegetable fatty
acids, water, salts or electrolytes, such as protamine sulfate,
disodium hydrogen phosphate, potassium hydrogen phosphate, sodium
chloride, zinc salts, colloidal silica, magnesium trisilicate,
polyvinyl pyrrolidone, cellulose-based substances, polyethylene
glycol (PEG; e.g., PEG400), sodium carboxymethylcellulose,
polyacrylates, waxes, polyethylene-polyoxypropylene-block polymers,
and wool fat.
[0039] In some cases, when a composition containing one or more
senotherapeutic agents (e.g., dasatinib and/or quercetin) is
administered to a mammal having, or at risk of developing, an
obesity-induced neuropsychiatric disorder (e.g., obesity-induced
anxiety), the composition can be designed for oral or parenteral
(including subcutaneous, intramuscular, intravenous, and
intradermal) administration to the mammal. Compositions suitable
for oral administration include, without limitation, liquids,
tablets, capsules, pills, powders, gels, and granules. Compositions
suitable for parenteral administration include, without limitation,
aqueous and non-aqueous sterile injection solutions that can
contain anti-oxidants, buffers, bacteriostats, and solutes that
render the formulation isotonic with the blood of the intended
recipient.
[0040] A composition containing one or more senotherapeutic agents
(e.g., dasatinib and/or quercetin) can be administered to a mammal
having, or at risk of developing, an obesity-induced
neuropsychiatric disorder (e.g., obesity-induced anxiety) in any
appropriate amount (e.g., dose). Effective amounts can vary
depending on the route of administration, the age and general
health condition of the subject, excipient usage, the possibility
of co-usage with other therapeutic treatments such as use of other
agents, and the judgment of the treating physician. An effective
amount of a composition containing one or more senotherapeutic
agents can be any amount that can treat a mammal having, or at risk
of developing, an obesity-induced neuropsychiatric disorder without
producing significant toxicity to the mammal. For example, an
effective amount of dasatinib (D) can be from about 1 milligram per
kilogram body weight (mg/kg) to about 20 mg/kg (e.g., about 5
mg/kg). For example, an effective amount of quercetin (Q) can be
from about 10 mg/kg to about 200 mg/kg (e.g., about 50 mg/kg). The
effective amount can remain constant or can be adjusted as a
sliding scale or variable dose depending on the mammal's response
to treatment. Various factors can influence the actual effective
amount used for a particular application. For example, the
frequency of administration, duration of treatment, use of multiple
treatment agents, route of administration, and severity of the
obesity-induced neuropsychiatric disorder in the mammal being
treated may require an increase or decrease in the actual effective
amount of senotherapeutic agent(s) administered.
[0041] A composition containing one or more senotherapeutic agents
(e.g., dasatinib and/or quercetin) can be administered to a mammal
having, or at risk of developing, an obesity-induced
neuropsychiatric disorder (e.g., obesity-induced anxiety) in any
appropriate frequency. The frequency of administration can be any
frequency that can treat a mammal having, or at risk of developing,
an obesity-induced neuropsychiatric disorder without producing
significant toxicity to the mammal. For example, the frequency of
administration can be from about twice a day to about once every 6
months, from about once a day to about once a week, or from about
once a week to about once every 6 months. In some cases, a
composition containing one or more senotherapeutic agents can be
administered once a day. The frequency of administration can remain
constant or can be variable during the duration of treatment. As
with the effective amount, various factors can influence the actual
frequency of administration used for a particular application. For
example, the effective amount, duration of treatment, use of
multiple treatment agents, and route of administration may require
an increase or decrease in administration frequency.
[0042] A composition containing one or more senotherapeutic agents
(e.g., dasatinib and/or quercetin) can be administered to a mammal
having, or at risk of developing, an obesity-induced
neuropsychiatric disorder (e.g., obesity-induced anxiety) for any
appropriate duration. An effective duration for administering or
using a composition containing one or more senotherapeutic agents
can be any duration that can treat a mammal having, or at risk of
developing, an obesity-induced neuropsychiatric disorder without
producing significant toxicity to the mammal. For example, the
effective duration can vary from several days, to several weeks, to
several months, or to a lifetime. In some cases, the effective
duration can range in duration from about several months to about
10 years. Multiple factors can influence the actual effective
duration used for a particular treatment. For example, an effective
duration can vary with the frequency of administration, effective
amount, use of multiple treatment agents, and route of
administration.
[0043] In certain instances, a course of treatment can be
monitored. In some cases, methods described herein also can include
monitoring the severity of an obesity-induced neuropsychiatric
disorder (e.g., obesity-induced anxiety) in a mammal. Any
appropriate method can be used to monitor the severity of an
obesity-induced neuropsychiatric disorder in a mammal. In some
cases, methods described herein also can include monitoring a
mammal being treated as described herein for toxicity. The level of
toxicity, if any, can be determined by assessing a mammal's
clinical signs and symptoms before and after administering a known
amount of a particular composition. It is noted that the effective
amount of a particular composition administered to a mammal can be
adjusted according to a desired outcome as well as the mammal's
response and level of toxicity.
[0044] The invention will be further described in the following
examples, which do not limit the scope of the invention described
in the claims.
EXAMPLES
Example 1: Obesity Induced Cellular Senescence Drives Anxiety-Like
Behavior Via Impaired Neurogenesis
[0045] This example shows that during obesity, glial cells show
increased markers of cellular senescence in the periventricular
region of the lateral ventricle (LV), a region in close proximity
to the neurogenic niche. Senescent glial cells in obese mice show
excessive fat accumulation, a phenotype termed accumulation of
lipids in senescence (ALISE). Importantly, specific clearance of
senescent cells alleviates the obesity-related impairment in adult
neurogenesis, and decreases obesity-induced anxiety-like behavior.
This work suggests that targeting senescent cells can be used as a
therapeutic avenue for treating obesity-induced anxiety.
Results
Obese Mice Show Increased Anxiety-Like Behavior not Related to Body
Mass
[0046] In order to investigate the relationship between obesity and
anxiety, eight month old C57Bl/6 mice were fed a high fat (60% of
calories from fat) or standard chow diet for 2 months. It was found
that body weight and body fat content were increased in high fat
diet (HFD) mice in comparison to chow-fed controls (FIG. 2A, B). To
measure anxiety-like behavior, the open-field (OF) test was
employed. This test evaluates the tendency of mice to remain close
to the walls and avoid open spaces (central zone), a phenomenon
known as thigmotaxis, which is widely used as an indication of
anxiety-like behavior (Simon et al., Behav Brain Res 61:59-64
(1994)). HFD fed mice were less inclined to explore the central
area of the open-field test chamber than the peripheral zone (FIG.
1A-C; FIG. 2D) and likewise the total distance covered was
significantly decreased in HFD animals during the test (FIG. 2C).
In order to account for the decreased activity in obese animals,
anxiety measurements were analyzed as a function of the total
distance travelled during experimental testing (FIG. 1B, D). It was
next investigated if body weight and body composition alone could
explain the observed anxiety-like behavior. Linear regression
analysis revealed no significant correlation between body weight or
% of fat mass on anxiety-like behavior in HFD fed mice (FIG. 1D, E;
FIG. 2E-J). This indicates that while weight gain is associated
with the onset of anxiety-like behavior, if a certain weight is
reached, no correlation between weight and anxiety is found, which
suggests that other factors apart from weight gain must play a
role.
[0047] As an additional measurement of anxiety-like behavior, the
elevated plus maze (EPM) test was used. The EPM is based on the
animal's natural fear of heights and open spaces. Increased
anxiety-like behavior in the EPM test is manifested as a decrease
in the number of head-pokes and entries into the open arms. It was
found that animals on a HFD had decreased entries into the open
arms of the EPM (frequency and time) compared to lean animals (FIG.
1F-H), which is indicative of an increased anxiety-like behavior.
Similarly to the OF test, there were no significant correlations
between body and fat mass and anxiety parameters in both lean and
HFD animals (FIG. 1I, J; FIG. 2G, H).
Pharmacogenetic and Pharmacologic Clearance of Senescent Cells
Alleviates Obesity-Related Behavioral Changes
[0048] It was investigated if senescent cells could contribute to
anxiety-like behavior during obesity by using the INK-ATTAC mouse
model, which allows the induction of suicide gene-mediated ablation
of p16.sup.Ink4a-expressing senescent cells upon administration of
the drug AP20187 (AP) (Baker et al., Nature 479:232-236 (2011); Xu
et al., Elife 4:e12997 (2015)).
[0049] Chow- and HFD-fed 10 month old mice were repeatedly treated
with AP or vehicle (FIG. 3A) over the duration of 10 weeks, which
resulted in no significant changes in body weight (FIG. 4A) or body
composition (not shown). To measure anxiety-like behavior, the OF
test was used and a previous observation that animals on HFD were
less inclined to explore the center of the open field chamber than
the periphery as measured by distance travelled (FIG. 3C) and
entries (FIG. 3D) to the central zone was confirmed. Furthermore,
mice on HFD travelled significantly less throughout the duration of
the tests, covering a smaller total distance (FIG. 4C). To take
this into account, all measured parameters were expressed as a
function of the total distance travelled. Clearance of
p16.sup.Ink4a-positive cells with AP reduced HFD-induced
anxiety-like behavior as measured by distance covered in the
central zone (FIG. 3B, FIG. 4B) and entries into the central zone
(FIG. 3C). However, AP treatment did not affect the total distance
travelled (FIG. 4C) or any of aforementioned parameters in chow-fed
mice (FIG. 3B-D).
[0050] Next, anxiety-like behavior was assayed with the elevated
plus maze (EPM). As previously observed, obese animals avoided
entries into the open arms of the EPM (frequency and time) compared
to lean animals (FIG. 3E-G and FIG. 4D, E), indicating increased
anxiety-like behavior. Consistent with the data from the OF test,
AP treatment significantly decreased the anxiety-like phenotype in
obese animals as indicated by the frequency of head entries into
the open arms of the EPM (FIG. 3E-G and FIG. 4D, E). Other
cognitive functions, such as short-term memory (FIG. 4F, G),
measured by the Stones maze test, were not altered by HFD or AP
treatment. Altogether, these results show that specific elimination
of p16.sup.Ink4a+-senescent cells from obese INK-ATTAC mice
alleviates anxiety-like behavior, but has no effect on memory
performance.
[0051] To exclude off-target effects of the drug AP, wild-type
C57Bl/6 mice were treated with the drug and tested for anxiety-like
behavior. Wild-type mice showed a significant difference between
chow and HFD in the OF test before the start of the treatment (FIG.
4H), but no difference was observed in HFD fed mice after treatment
with AP (FIG. 4I).
[0052] In addition to HFD fed mice, complementary experiments were
conducted in db/db mice in which obesity is caused by a point
mutation in the leptin receptor gene lepr, leading to spontaneous
type 2 diabetes (Wang et al., Current Diabetes Reviews 10:131-145
(2014)). These mice were treated intermittently for two months with
the senolytic drug cocktail, Dasatinib and Quercetin (D+Q) (Zhu et
al., Aging Cell 14:644-658 (2015)). Db/db mice have significantly
increased body weights and adipose depot weights when compared to
lean db.sup.+/- heterozygous littermates, but interestingly body
weight did not change over the course of D+Q treatment (FIG. 4J,
K).
[0053] Similarly to HFD fed mice, db/db mice exhibited increased
anxiety-like behavior as assessed by the OF test (FIG. 3H-J). It
was observed that the total distance covered (FIG. 3I) and the
number of entries (FIG. 3J) into the central zone were
significantly reduced in db/db mice compared to their non-obese,
db.sup.+/- heterozygous littermates, a phenotype which could be
significantly alleviated by treatment with senolytic compounds
Dasatinib and Quercetin (D+Q). Obese db/db mice covered a
significantly shorter distance in comparison to lean littermates,
however D+Q treatment did not change total distance covered in
db/db or db/db.sup.+/- mice (FIG. 4L).
[0054] Finally, these results were confirmed in a cohort of
double-transgenic, INK-ATTAC;db/db mice. Similarly to treatment
with D+Q, genetic clearance of p16.sup.Ink4a-positive senescent
cells in INK-ATTAC;db/db mice did not alter body weight, body
composition, or activity (FIG. 4M-O). Importantly, clearance of
p16.sup.Ink4a-positive senescent cells in INK-ATTAC;db/db mice
significantly reduced anxiety-like behavior as assayed by OF test
(FIG. 3K, L). To exclude off-target effects of the drug AP, db/db
mice were treated with the drug. No off-target effects of the AP
drug were observed on anxiety-like behavior by OF test (FIG. 4P).
Linear regression analysis showed no significant correlations
between body weight and anxiety-like behavior in HFD fed INK-ATTAC
(with and without AP) or db/db (with or without AP or D+Q) (FIG.
4Q-S).
[0055] These data show that pharmacological or pharmacogenetic
clearance of senescent cells in two different models of obesity
significantly alleviates anxiety-like behavior.
Pharmacological and Pharmacogenetic Senolytic Approaches Reduce
Senescent Cell Burden and Alleviate Systemic Inflammation
[0056] To investigate the effectiveness of senescent cell-clearance
in HFD mice, senescent markers were measured in the perigonadal
adipose tissue, a tissue previously shown to exhibit a marked
increase in the number senescent cells with age (Schafer et al.,
Nature Commun. 8:14532 (2017); Tchkonia et al., Aging Cell
9:667-684 (2010); Xu et al., Elife 4:e12997 (2015)).
[0057] It was found that the senescence markers SA-.beta.-Gal,
p16.sup.Ink4a, and telomere-associated DNA damage foci (TAF) were
increased in INK-ATTAC mice on HFD and were significantly reduced
upon administration of AP (FIG. 5A-C). p21 and .gamma.-H2A.X were
increased in HFD animals, but were not significantly changed upon
AP treatment (FIG. 6A, B). Similarly, it was found that db/db mice
had an increased senescent cell burden (measured by SA-.beta.-Gal
and TAF frequency) compared to db/db' mice in the perigonadal fat,
which was significantly reduced by treatment with the senolytic
cocktail D+Q (FIG. 5D-F).
[0058] Given that the senolytic approaches applied act
systemically, it is possible that they reduce SASP factors which
can penetrate the blood-brain-barrier and therefore impact on the
brain. To investigate that, blood plasma was analyzed for a large
array of SASP factors.
[0059] Evaluation of circulating cytokines in the bloodstream of
chow- and HFD-fed INK-ATTAC mice revealed that HFD resulted in the
up-regulation of known SASP factors such as G-Csf, Il-1.beta.,
Kc/Cxcl1, Mcp-1, Mig, and Tnf-.alpha. which were down-regulated
upon AP treatment (FIG. 5G). Similarly, SASP factors in the blood
plasma of db/db mice were increased in comparison to lean db/+
littermates and reduced after treatment with D+Q (FIG. 5H). The
expression of cytokines in the bloodstream was correlated with
parameters of anxiety-like behavior measured in OF test.
Significant negative correlations were observed between the plasma
levels of Cxcl1, G-Csf, and Mig and different anxiety-like
measurements in AP-treated HF mice (FIG. 5I-K and FIG. 6C) and
D+Q-treated db/db mice (FIG. 6D, E), whereas no correlation was
found for Tnf-.alpha., Il-6, and Mcp-1 (FIG. 6F-H). These results
led to further investigation of the impact of systemic factors on
the observed anxiety phenotype. Lean animals were injected with
Cxcl1. Injection of Cxcl1 lead to increased levels of circulating
Cxcl1 (FIG. 6I). Additionally, increased plasma Cxcl1 decreased
slightly the body weight, but did not change body composition (FIG.
6J, K). Examination of anxiety-like behavior using the OF (FIG. 6L)
and EPM (FIG. 6M) tests showed no difference between treated and
non-treated animals. To further examine the role of Cxcl1 in
anxiety-like behavior, HFD mice were treated with Reparixin, which
inhibits Cxcl1 receptors Cxcr1 and Cxcr2. Mice on Reparixin showed
a small increase in body weight (FIG. 6N) but no difference in body
fat (FIG. 6O) in comparison to non-treated mice. Again no
difference was observed in behavior between the treated and
non-treated groups, when animals were tested in the OF box (FIG.
6P) or the EPM (FIG. 6Q). These results suggest that Cxcl1 alone is
not sufficient to induce an anxiety-like phenotype, however, it
does not exclude the possibility that other soluble SASP factors
are involved in the process.
[0060] Recently, it has been shown that transplantation of
relatively low numbers of senescent cells in young animals resulted
in physical dysfunction measured by Rotarod performance, grip
strength, or endurance when compared to transplantation of young
cells (Xu et al., Nature Medicine 24:1246-1256 (2018)). This study
showed that transplantation of senescent cells resulted in
long-lasting systemic effects in tissues located distantly from
where senescent cells were injected.
[0061] To test if senescent cells could induce anxiety-like
behavior via systemic effects, young or senescent cells were
transplanted into lean mice and assessed behavior and physical
function 6 and 12 weeks later. It was confirmed the previous
observations that transplanted senescent cells reduced physical
function, as measured by Rotarod (FIG. 6J), but had no effect on
anxiety-like behavior using the OF test (FIG. 6T-W). Together,
these experiments suggest that presence of senescent cells
elsewhere in the body are not sufficient to induce an anxiety-like
phenotype in mice.
Senolytic Treatment Reduces the Frequency of Senescent Cells in
Amygdala and Hypothalamus but not Other Regions of the Brain
[0062] It was next examined if obesity could induce senescence
specifically in the brain, thereby contributing to anxiety. Markers
of senescence were first assessed in these regions of the brains of
obese and lean INK-ATTAC mice treated with and without AP. No
differences were found in the senescent markers p21, p16,
.gamma.-H2A.X, and TAF between any of the experimental groups (FIG.
8A-D). This correlates with the absence of differences in memory
and learning in any of the experimental groups as assessed by the
Stone's maze (FIG. 4F, G).
[0063] Interestingly, assessment of senescent cells in the
amygdala, a brain region associated with emotional responses
including anxiety and fear (Adhikari et al., Nature 527:179-185
(2015)), exhibited a significant increase in the number of
p16.sup.Ink4a-positive cells in HFD-fed mice (FIG. 7A). Analysis of
TAF positive neurons in the basomedial layer of the amygdala showed
a significant increase in HFD mice and a significant decrease after
treatment with AP (FIG. 7A-D). Next, the abundance of senescent
cells in the hypothalamus close to the 3.sup.rd ventricle was
analyzed and it was found that NeuN.sup.neg (FIG. 7E, F) and
NeuN.sup.pos (FIG. 8E, F) cells show a significant increase in HFD
mice compared to lean mice and a significant decrease after
treatment with AP.
[0064] Together, these data indicate that HFD does not induce
senescence in regions of the brain implicated in learning, memory,
and motor-neuron control such as the cortex, cerebellum, and
hippocampus. However, HFD induces senescence in the hypothalamus
and amygdala, which may contribute to its effects on anxiety-like
behavior and treatment with AP reduced senescent cell abundance and
attenuated these behavioral changes.
Clearance of Senescent Cells Decreases Periventricular Accumulation
of Lipid-Laden Glia in Obese Animals
[0065] A connection between senescence and fat accumulating in the
brain was investigated.
[0066] Analysis of Perilipin 2 (Plin2) expression (a protein which
surrounds lipid droplets) in the brain of HF diet mice revealed a
significant increase in Plin2.sup.+ cells (FIG. 10A) located in
close proximity to the lateral ventricle (LV) compared to chow-fed
controls (FIGS. 9A, B). Plin2.sup.+ cells were also detected around
the 3.sup.rd and 4.sup.th ventricles and the periaqueductal gray
(PAG) matter (FIG. 10B) but not in other brain regions.
Double-staining for Plin2 and the cell type-specific markers,
vimentin (Vim), Iba1, and NeuN, indicated that Plin2.sup.+ cells
are mostly astrocytes (41%) and microglia (19%) (FIG. 9C, D).
[0067] In order to investigate if Plin2.sup.+ cells show features
of senescence, the senescence marker TAF was analyzed in
combination with immunostaining against Plin2. Higher mean values
and higher frequencies of TAF in Plin2.sup.+ cells (FIG. 9E) were
found, while total DNA damage did not change (FIG. 10C). Together,
these findings indicate that a HFD contributes to increased numbers
of senescent cells in the periventricular region of the brain and
that these cells preferentially accumulate fat. Given that a
similar phenomenon was observed in senescent hepatocytes and
fibroblasts (see, e.g., Ogrodnik et al., Nat Commun. 8:15691
(2017)), this phenotype was termed Accumulation of Lipids in
Senescence (ALISE).
[0068] To further investigate the impact of senescent cells on the
build-up of fat in the brain, the INK-ATTAC mouse model (Baker et
al., Elife. 4:e12997 (2015); and Baker et al., Nature 479:232-236
(2011)) was used. Treatment of HFD INK-ATTAC mice with AP resulted
in a significant reduction of Plin2.sup.+ cells (FIG. 9F, G), as
well as cells bearing senescent markers (FIG. 9H). Further, the
frequency of cells containing lipid droplets and the senescent
marker TAF in the LV of db/db animals was analyzed, and it was
found that these were significantly increased in db/db in
comparison to db/db.sup.-/+ animals and significantly reduced after
treatment with the senolytic cocktail D+Q (FIG. 10D-F).
Additionally, neuroinflammation was assessed by conducting RNA-In
situ hybridization against SASP factors Cxcl1 and Il-6 in
combination with immunostaining for Plin2 in the LV of HFD mice
(FIG. 9I). Interestingly, the majority of Plin2.sup.+ cells were
also positive for Cxcl1 and Il-6 (FIG. 9J, K).
[0069] Lastly, anxiety markers in HFD animals, such as distance
travelled in the central zone and entries into the central zone,
showed a strong negative correlation with the abundance of
Plin2.sup.+ cells detected in the lateral ventricle (FIG. 9J, K).
Together, these data support a causal link between the accumulation
of lipid-laden senescent glial cells in obese animals and
anxiety-like behavior.
Suppression of the ALISE Phenotype Reduces Accumulation of
Cytosolic Chromatin Fragments (CCF) and the SASP
[0070] To further investigate the impact of fat accumulation on
cell senescence, mouse adult fibroblasts (MAF) were used and
senescence was induced by X-ray irradiation as described elsewhere
(see, e.g., Jurk et al., Nat Commun 2 (2014); Ogrodnik et al., Nat
Commun. 8:1569 (2017)). Senescent cells were cultured in the
presence or absence of external sources of lipids. It was found
that in the absence of extracellular lipids, the ALISE phenotype
cells (assessed by lipophilic dye, Nile Red) was suppressed (FIG.
11A, B). Next, it was investigated if the impact of fat build-up on
different markers of cellular senescence (FIG. 11C-K; FIG. 12A).
Recently, it has been reported that senescent cells contain
cytoplasmic chromatin fragments (CCF) (Ivanov et al., Journal of
Cell Biology 202:129-143 (2013)), which activate the DNA-sensing
cGAS-STING pathway which is a major driver of the SASP (Dou et al.,
Nature 550:402 (2017)). Interestingly, it was found that abrogation
of the ALISE phenotype significantly reduced CCF in senescent cells
(FIG. 11C, D), but not the average number of DNA damage foci (FIG.
11E, F) or bi-nuclearity (FIG. 12A). Consistent with the hypothesis
that enhanced lipid deposition impacts on CCF and the SASP, it was
found that depriving cells of lipids resulted in a drastic
reduction of several key components of the SASP, such as Il-6, Kc
(Cxcl-1), Ip-10 (Cxcl-10), and Lix (Cxcl-5) (FIG. 11G-K). To
investigate if these findings were restricted to MAFs, similar
experiments were conducted in primary mouse astrocytes. Similarly
to MAFs increased build-up of fat, increased TAF, and higher
numbers of CCFs were found in senescent astrocytes (FIG.
11L-O).
[0071] These data show that excessive lipid accumulation during
senescence (ALISE) may be a contributor of genomic instability,
resulting in release of chromatin fragments and activation of the
SASP.
Impaired Neurogenesis in HF Animals is Rescued by Clearance of
Senescent Cells
[0072] In HFD mice, it was observed that Plin2.sup.+ senescent
glial cells are frequently found in close proximity to cells
expressing doublecortin (Dcx), a marker of neuronal precursor cells
and immature neurons (FIG. 14A). Thus, the presence of ALISE glial
cells in the subventricular zone (SVZ) was investigated.
[0073] Lean and obese INK-ATTAC mice were treated with or without
AP as previously described. Following organ harvesting, single-cell
suspensions were obtained from one brain hemisphere and analyzed
them by Cytometry by Time Of Flight (CyTOF), which allows mapping
and discriminating between different brain cells including
astrocytes, oligodendrocytes, microglia, neurons, ependymal cells,
pericytes, and endothelial cells. The second brain hemisphere was
reserved for histological analyses (FIG. 13A, B).
[0074] It was found that brains of mice fed a HFD did not exhibit
significant changes in the frequencies of oligodendrocytes
(CNPase.sup.+ or OSP.sup.+), microglia (CD11b.sup.+, CD45), mature
neurons (NeuN.sup.+), or endothelial cells (CD31.sup.+ or
CD146.sup.+) (FIG. 13C; FIG. 14B-M). However, populations of
neuronal precursor cells (Nestin.sup.+), immature neurons
(Dcx.sup.+), and ependymal cells (CD133.sup.+) were significantly
decreased in animals subjected to HFD feeding (FIG. 13C-G).
Clearance of senescent cells by AP did not alter the frequencies of
oligodendrocytes (CNPase.sup.+ or OSP.sup.+), microglia
(CD11b.sup.+, CD45.sup.-), mature neurons (NeuN.sup.+), or
endothelial cells (CD146.sup.+) (FIG. 13C; FIG. 14B-M), but
significantly increased neuronal precursor cells, immature neurons,
and ependymal cells (FIG. 13C-G). AP treatment was sufficient to
induce partial recovery of obesity-related stem cell depletion
(FIG. 13C, E) and to replenish CD133.sup.+ and Nestin.sup.+ cell
abundance (FIG. 13F, G). Analysis of cell populations in brains of
db/db mice showed a similar pattern to HFD-fed mice. Markers for
immature neurons (Dcx.sup.+), ependymal cells (CD133.sup.+), and
neuronal precursor cells (Nestin.sup.+) were all significantly
upregulated after senolytic treatment (FIG. 14N-P).
[0075] These findings were validated by performing immunostaining
for Dcx (FIG. 13H, I) in the olfactory bulb and EdU-pulse labelling
(FIG. 14S, T) in the subventricular zone of HFD animals. Analysis
of Dcx.sup.+ cells in the olfactory bulb showed a positive
correlation between the area occupied by Dcx.sup.+ cells in the
granular cell layer of the olfactory bulb and the frequency of
Dcx.sup.+ cells detected by CyTOF (FIG. 13J). Quantification of
Dcx.sup.+ cells (FIG. 14U, V) and EdU.sup.+ cells (FIG. 14W) in the
subgranular zone (SGZ) of the hippocampus did not correlate with
results from CyTOF, implying that adult neurogenesis in SVZ, but
not in the SGZ, is affected by the presence of senescent cells
induced by obesity. Interestingly, clearance of these senescent
cells led to a significant increase in the population of astrocytes
(FIG. 14G-I) in obese animals, whereas no differences between lean
and obese animals were detected. This finding was further confirmed
by immunostaining for Gfap (FIG. 14Q, R). Finally, it was found
that frequencies of Plin2.sup.+ glia correlated with markers of
ependymal cells and markers of adult neurogenesis Nestin.sup.+
(FIG. 13K) and Dcx.sup.+ (FIG. 13L). Similarly, individual
differences in distance travelled in the central area were
positively correlated with frequencies of Dcx.sup.+ and
Nestin.sup.+ cells (FIG. 13M, N).
[0076] In summary, these data indicate that senescent cells play a
causal role in the decreased neurogenesis induced by HFD. Targeting
senescent cells in obese mice alleviates obesity-related
anxiety-like behavior related to clearance of periventricular fat
accumulation and restoration of adult neurogenesis.
Methods
Animals
[0077] Experimental procedures were approved by the Institutional
Animal Care and Use Committee at Mayo Clinic (protocol A26415).
INK-ATTAC.sup.+/- transgenic mice were generated and genotyped as
described elsewhere (see, e.g., Baker et al., Nature 479:232-236
(2011)). Briefly, INK-ATTAC mice were produced and phenotyped at
Mayo Clinic. Controls for the INK-ATTAC experiments were
INK-ATTAC-null C57BL/6 background mice raised in parallel. C57BL/6
db/db and db/- mice were purchased from Jackson Laboratories.
[0078] Mice were housed 2-5 mice per cage, at 22+/-0.5.degree. C.
on a 12-12 hour day-night cycle and provided with food and water ad
libitum. For high fat diet-induced obesity studies, mice were
randomly assigned to chow or high fat diet groups. Mice were fed
the high fat diet for 2-4 months before experiments started. High
fat food was purchased from Research Diets (cat no #D12492). 60% of
calories in this high-fat diet are from fat. Standard mouse chow
diet was obtained from Lab Diet (cat no #5053).
[0079] INK-ATTAC mice were injected intraperitoneally (i.p.) with
AP20187 (10 mg/kg) or vehicle for 3 days every 2 weeks for a total
of 8-10 weeks.
[0080] Senolytic-treated db/db mice were gavaged with Dasatinib (D;
5 mg/kg) and quercetin (Q; 50 mg/kg) or vehicle for 5 days every 2
weeks for 8 weeks.
[0081] For off target effect measurements db/db and HDF mice (fed
with HFD for 2 months prior treatment) were injected
intraperitoneally (i.p.) with AP21087 at 10 mg/kg or vehicle for 3
days every 2 weeks for 8 weeks.
[0082] Recombinant CXCL1 (Peprotech, #250-11) or vehicle (PBS) was
administered to lean C57BL/6 via i.p. injection (5 .mu.g/kg in PBS)
daily for 7 days. 2 hours after the last injection mice were tested
in open field and elevated plus maze and afterwards dissected.
[0083] Reparixin L-lysine salt (MedChemExpress, #HY-15252) or
L-Lysine hydrochloride (MedChemExpress, #HY-N0470) was dissolved in
H.sub.2O was administered to obese C57BL/6 mice (fed for 2 months
with high-fat diet) via subcutaneous injection (30 mg/kg) twice per
day for 2 weeks. 2 hours after the last injection mice were tested
in open field and elevated plus maze and afterwards dissected.
[0084] Tissues from mice sacrificed at the indicated time points
were snap-frozen in liquid nitrogen for biochemical studies or
fixed in 4% PFA for 24 hours prior to processing and paraffin
embedding. Paraffin-embedded tissues were cut at 3 .mu.m or 10
.mu.m intervals.
Transplantation
[0085] Wild-type C57BL/6 mice were obtained from the National
Institute on Aging (NIA) and maintained in a pathogen-free facility
at 23-24.degree. C. under a 12 hours light, 12 hours dark regimen
with food and water ad libitum. Cell transplantation was done as
previously described (Xu et al., Nature Medicine 24:1246-1256
(2018)). Briefly, when mice were 18 months of age, they were
anesthetized using isoflurane and were injected intraperitoneally
with 150 .mu.l PBS through a 22-G needle, containing 10.sup.6
control or senescent mouse preadipocytes cells, or only PBS.
Preadipocytes were obtained from inguinal fat from young Luciferase
transgenic C57BL/6 mice from The Jackson Laboratory (Bar Harbor,
Me.; stock no. 025854). Senescence was induced by 10 Gy of cesium
radiation. Open field testing was carried out at 2 and 6 weeks
after transplantation and Rotarod performance was tested 2 and 12
weeks after transplantation.
Body Composition
[0086] Lean and fat mass of individual mice were determined by
quantitative nuclear magnetic resonance using an EchoMRT analyser
(Houston, Tex.) and expressed as a function of body weight.
Un-anesthetized animals were placed in a plastic tube that was
introduced into the EchoMRT instrument. Body composition,
comprising fat mass and lean mass, was determined in approximately
90 seconds per animal.
Open Field Testing
[0087] Locomotor activity and anxiety-like behavior of mice were
assessed in sound-insulated, rectangular activity chambers (Med
Associates Inc., St Albans, Vt., USA: W.times.L.times.D=27
cm.times.27 cm.times.20 cm with continually running fans, infrared
lasers, and sensors). Beam breaks were assessed in 2-minute bins
over 30 minutes, converted automatically to current mouse location
and distance travelled (cm), and recorded on a computer with Med-PC
software Version 4.0. Before the test, mice were acclimatized to
the room for 1-1.5 hours before being introduced into the chambers.
Mice were habituated for 5 minutes in the Open Field chamber
(without recording) then placed for another 5 minutes in the home
cage. Afterwards, mice were introduced back to the chambers and all
mouse movements were recorded for 30 minutes. Anxiety was
quantified by the distance mice travelled in the central 25% of the
chamber (zone 1) as a function of the total distance mice travelled
and by frequencies of entries into zone 1.
Elevated Plus Maze
[0088] A grey colored elevated plus maze apparatus was used. Two
open arms (25.times.5 cm) and two closed arms (25.times.5 cm) were
attached at right angles to a central platform (5.times.5 cm). The
apparatus was set 40 cm above the floor. Mice were first
acclimatized to the room for 1-1.5 hours. Mice were then placed
individually on the central platform with their back to one of the
open arms. Before the test, mice were habituated for 1 minute to
the maze, then placed back in the home cage for 5 minutes. Mice
were tested for 5 minutes during which they could freely explore
the apparatus. Tracking software (Ethovision) recognizes mouse
head, central body point, and the base of the tail. Anxiety was
quantified by frequency of and time spent during head pokes/dips
toward open arms. Higher anxiety is indicated by a lower frequency
of movement into open arms and less time spent there.
Rotarod
[0089] Rotarod performance test evaluates mouse balance and motor
coordination. Mice were brought to the test room a day before
testing and habituated overnight. For the baseline tests, mice were
trained on Rotarod (3375-M5; TSE systems) first for three
consecutive days. Mice were placed (having their back turned
towards the experimenter) on the rotating rod of 4.0 cm diameter.
Mice trained to stay on the rod for 200 seconds at one constant
speed per day, incrementing speed each day from 4 rpm, 6 rpm, and 8
rpm. If a mouse fell during training, it was put back on the rod.
For the test on the fourth day, the Rotarod started at 4 rpm and
steadily accelerates to 40 rpm over a 5 minute interval. The speed
at which mice dropped was recorded, in four consecutive trials. 2
and 12 weeks after baseline measurements mice were tested again,
habituating overnight prior test day. The average was normalized to
the baseline and taken as an indicator of mouse balance and motor
coordination.
Stone's T-Maze
[0090] A water-motivated version of the Stone's T-maze was used to
measure parameters of cognition. A straight run (for pre-training)
or Stone's T-maze were placed into a steel pan filled with water to
a depth of approximately 3 cm so that half the height of the
interior walls of the maze were under water. The ceilings of both
the straight run and maze were covered with clear acrylic to
prevent mice from rearing out of the water. These dimensions
created a situation that enables the mice to maintain contact with
the floor while keeping their heads above water. The mice were
placed into a start box and were pushed into the maze using a
sliding panel. At the end of the straight run or maze there was a
goal box that contains a ramp to a dry floor, which allows the mice
to escape from the water upon successful completion of the straight
run or maze. On day one, mice underwent straight run training to
establish the concept that moving forward allows them to escape the
water by reaching a water-free goal box. Successful completion of
this phase requires the mice to reach the goal box in 10 seconds or
faster in 8 out of 10 trials. Mice that did not reach this
criterion were excluded from further testing. Maze training
commenced the following day. Mice had to complete 9 maze
acquisition trials in a single day. All mice per group performed
one trial before performing the next one. Runs using between 6 and
8 mice resulted in inter-trial intervals (ITI) of approximately
5-12 minutes. During ITI, mice were placed in a holding cage
containing a dry towel that was additionally heated by a red heat
lamp. Primary measures of learning and memory were the latency to
reach the goal box and the numbers of errors committed. An error
was defined as complete entry of the mouse's head or the whole body
into an incorrect path. During the acquisition phase, if any mouse
failed to reach the goal box within 5 minutes, the trial was
terminated and scored as a failure. Any mouse having 3 failures was
removed from further trials. No mouse was excluded from this
study.
RT-PCR
[0091] Total RNA was extracted from white adipose tissue and brain
using Trizol (Life Technologies, Carlsbad, Calif.) and reverse
transcribed to cDNA with a M-MLV Reverse Transcriptase kit (Life
Technologies). Real-time PCR was performed in a 7500 Fast Real Time
PCR System (Applied Biosystems, Foster City, Calif.) using TaqMan
Fast Universal PCR Master Mix (Life Technologies) and predesigned
primers and probes from Applied Biosystems (Assay ID: Mm00494449_m1
[CDKN2A]; Mm04205640_g1 [CDKN1A]; Mm00446191_m1 [IL6]). Target gene
expression was expressed as 2-.DELTA..DELTA.CT by the comparative
CT method and normalized to the expression of TATA-binding protein
(TBP) (Assay ID: Mm01277042 ml [TBP]).
Cellular Senescence-Associated Beta-Galactosidase (SA-.beta.-Gal)
Activity
[0092] On the day of the sacrifice, a small piece of adipose tissue
was fixed with 2% PFA and 0.5% glutaraldehyde (Sigma) for 15
minutes at room temperature before being incubated overnight in
SA-.beta.-Gal solution (150 mM NaCl (Sigma), 2 mM MgCl.sub.2
(Sigma), 40 mM Citric Acid (Sigma), 12 mM NaPO.sub.3 (Sigma), 400
.mu.g/ml X-gal (Thermofisher), 2.1 mg/ml potassium
hexacyanoferrat(II)trihydrate, and 1.65 mg/ml Potassium
hexacyanoferrat(III)trihydrate (Sigma), pH 6.0) at 37.degree. C.
overnight. Fat chunks were washed with PBS three times and stored
in PBS at 4.degree. C. protected from light. Within 3 days, adipose
tissue was stained with Hoechst solution (1:5000; Thermofisher),
lightly squashed between two 1.times.3 inch glass slides, and
imaged using a light microscope. 10-20 random visual fields were
captured at 20.times. magnification at light exposure identical for
all the samples. Images were quantified by manual counting of
SA-.beta.-Gal positive cells by a blinded assessor and the data
were expressed as percent of total DAPI-positive cells.
Mass Cytometry/CyTOF in Brain and Fat
[0093] This technique uniquely combines time-of-flight mass
spectrometry with metal-labelling technology to enable detection of
up to 40 protein targets per cell. A panel of antibodies based on
surface markers, transcription factors, and cytokines (see Table 1)
was designed for brain mass cytometry/cytometry by time of flight
(CyTOF). Each antibody was tagged with a rare metal isotope and its
function verified by mass cytometry according to the factory manual
(Multi Metal labelling Kits, Fluidigm, CA). A CyTOF-2 mass
cytometer (Fluidigm, South San Francisco, Calif.) was used for data
acquisition. Acquired data were normalized based on normalization
beads (Ce140, Eu151, Eu153, Ho165, and Lu175). A single brain
hemisphere was dissociated into a single-cell suspension using
brain tissue dissociation kits (Adult Brain Dissociation Kit,
Miltenyi Biotec Inc., CA). Collected cells were incubated with
metal-conjugated antibodies and, for testing intracellular
proteins, including transcription factors and cytokines, fixation
and permeabilization was conducted according to the manufacturer's
instructions (Transcription Factor Staining Buffer Set,
eBioscience, San Diego, Calif.). CyTOF data were analyzed by
Cytobank (Santa Clara, Calif.).
TABLE-US-00001 TABLE 1 Antibodies used in CyTOF Name of an Company
producing Catalogue Metal antigen antibody number Isotopes Dilution
CD11b (Mac-1) Fluidigm 3154006B 154Sm 1:200 CD45 Fluidigm 3089005B
89Y 1:400 nestin Abcam ab6142 148Nd 1:100 Dex Abcam ab135349 173Yb
1:100 vimentin Abcam ab8978 161Dy 1:100 CD133 Biolegend 141202
153Eu 1:100 ACSA-2 Miltenyi Biotec 130099138 142Nd 1:100 Gfap
ebioscience 14-9892-82 172Yb 1:100 CD31 Fluidigm 3165013B 165Ho
1:100 NeuN Abcam ab177487 169Tm 1:50 CD146 Fluidigm 3141016B 141Pr
1:100 CNPase Abcam ab53041 151Eu 1:200 OSP Abcam ab53041 159Tb
1:200
Cytokines
[0094] Serum levels of cytokines: Eotaxin, G-Csf, Tnf-.alpha.,
Il-6, Ifn-.gamma., Il-1.alpha., Il-1.beta., Il-17, Il-2, Kc/Cxcl1,
Mcp-1, M-Csf, Mig, Mip-1.alpha., and Mip-1.beta. were determined
using a Multiplexing LASER Bead Assay (Mouse Cytokine
Array/Chemokine Array 31-Plex (MD31), Eve Technologies; Canada).
Blood was withdrawn from mice by punctuation of the sub-mandibular
vein at the day of dissection before an animal was sacrificed. 50
.mu.L of serum were shipped to Eve Technologies on dry ice. Due to
high variability of data an unbiased elimination of outliers was
performed using ROUT's method (Graphpad 7 Prism). The same panel
was used to detect SASP in MAF and 50 .mu.l of media was shipped to
Eve Technologies on dry ice.
Cell Culture and Mouse Adult Fibroblasts (MAF)
[0095] MAF were extracted from 3-5 month old male C57BL/6 male
mice. Ear clippings were transported and stored (not longer than 1
hour) in serum-free DMEM on ice. Punches were washed three times
with serum-free media, finely cut, and incubated for 2-3 hours at
37.degree. C. in DMEM containing 2 mg/ml collagenase A. A
single-cell suspension was obtained by repeated pipetting and
passing through a 24-G fine needle. Cells were centrifuged for 10
min at 1,000 r.p.m. and cultured in Advanced D-MEM/F-12 (DMEM,
Invitrogen) plus 10% FBS (Sigma) in 3% 02 and 5% CO.sub.2. Each
cell strain was derived from a separate donor mouse and expanded
until enough cells are generated for freezing aliquots. For each
experiment, MAFs were defrosted, seeded and allowed to grow for 24
hours and then X-ray irradiated with 10 Gy using a PXI X-Rad 225
(RPS Services Ltd) to induce cellular senescence. Media were
changed twice a week. The last medium change was performed at day
20 after senescence induction (IR) and cells were fixed in 2% PFA
the next day.
[0096] For cytokine measurements media from the last 24 hours of
culture (before cell fixation) were sent to Eve Technologies for
SASP assessment (Mouse Cytokine Array/Chemokine Array 31-Plex
(MD31)).
Lipid Deprivation Experiments
[0097] Under normal conditions MAFs were kept in Advanced DMEM/F-12
(DMEM, Invitrogen) supplemented with 10% fetal bovine serum (FBS)
(Sigma), 100 IU/ml penicillin/streptomycin, and 2 mM L-glutamine.
In order to reduce content of lipids in tissue culture media,
lipid-deprived FBS (Biowest) was used. Therefore, media containing
standard FBS (with lipids) were designated "LIPID" and media
containing lipid-deprived FBS were designated as "NO LIPID". Young
(control) cells were kept for at least 7 days under "NO LIPID"
conditions before they were collected or senescence was
induced.
Mouse Neocortical Astrocytes
[0098] Astrocytes were extracted from 16-day-old embryo brains of
either sex. At 16th day of pregnancy mice were sacrificed and
brains of embryos were dissected. Neocortex was isolated and
homogenized by pipetting through a fire-polished, FBS-coated
Pasteur pipette. Bigger pieces of the neocortex were isolated by
sedimentation and supernatant was centrifuged to isolate
astrocytes. Astrocyte cultures were seeded at a density of
0.5.times.10.sup.6 cells/ml on culture dishes that had been coated
previously with 15 .mu.g/ml poly-1-ornithine overnight and
subsequently washed with H.sub.2O and PBS. Astrocytes were
maintained in DMEM/F12 medium supplemented with 5 mM HEPES, 33 mM
glucose, 13 mM sodium bicarbonate, 10% fetal bovine serum, 2 mM
glutamine 100 U/ml penicillin and 100 .mu.g/ml streptomycin (all
from Invitrogen). Cells were cultured at 37.degree. C. in a
humidified atmosphere of 5% CO.sub.2 and 3% 02. Induction of
senescence and assessment of senescence markers and the ALISE
phenotype were performed as for MAFs.
Immuno- and Nile Red Staining for MAFs and Astrocytes
[0099] MAFs and astrocytes were plated on 19 mm (diameter)
coverslips and at the end of experiments washed briefly with PBS
and fixed for 10 minutes with 2% paraformaldehyde dissolved in PBS.
Cells were permeabilized for 5 minutes with 0.5% TRITON X-100
dissolved in PBS. Cells were incubated with blocking buffer (5%
normal goat serum (S-1000, Vector Laboratories) in PBS) for 60
minutes at room temperature. Plin2 (PROGEN #GP46, 1:250) 53BP1
(Novus Biologicals, #NB100-304, 1:250) and GFAP (Synaptic Systems,
#173 004, 1:1000) antibodies were diluted in blocking buffer and
applied overnight at 4.degree. C. The next day, cells were washed
three times with PBS and incubated for 60 minutes with secondary
Alexa Fluor 594, goat, anti-guinea pig antibody (1:1000) for Plin 2
staining; Alexa Fluor, goat, anti-guinea pig (1:1000) for 53BP1
staining or Alexa Fluor, goat, anti-guinea pig (1:1000) for GFAP
staining. For quantification of senescence markers coverslips were
washed 3 times in PBS, then mounted in Vectashield, DAPI-containing
mounting media. For assessment of lipid accumulation cells were
washed 3 times with PBS before and after DAPI solution (PARTEC) was
added for 30 minutes at room temperature. 2 .mu.l of Nile red
solution (Nile red (Sigma N3013) 150 .mu.g ml.sup.-1 in acetone)
were added to 1 ml 80% glycerol (in Milli-Q water) and mixed
thoroughly. 20 .mu.l of Nile Red/glycerol were directly added to
each cell sample and mounted on a glass microscope slide. Images
were taken immediately after mounting using a Leica DM5500
widefield fluorescence microscope with a 20.times. objective lens.
Area of lipid droplets was quantified using ImageJ ("Analyze
particles" tool) in >50 cells in .gtoreq.10 images.
EdU Experiments
[0100] For EdU experiments, HFD and control chow-fed INK-ATTAC mice
treated with AP were injected with EdU (Life Technologies) at a
dose of 123 mg/kg with the final concentration of 6.15 mg/mL,
dissolved in sterile PBS (pH 7.4, Fisher Scientific), 2 hours
before perfusion. 15 minutes intervals were allowed between mice
injections to consider time needed for perfusing each mouse. The
animals were deeply anesthetized with 90 mg/kg ketamine and 10
mg/kg of xylazine in sterile PBS prior perfusion. Transcardiac
perfusion with PBS was followed by perfusion with 4%
paraformaldehyde in PBS chilled on ice. Brains were harvested and
postfixed overnight in 4% paraformaldehyde in PBS at 4.degree. C.,
washed with PBS, and stored at 4.degree. C. for vibratome
sectioning. Sagittal brains sections of 50 .mu.m were cut on a
vibratome and collected sequentially in 6 different plate wells,
total of 13 sections, 250 .mu.m apart in each well, representing
1/6 of the brain hemisphere. Sections were stained free-floating in
12 well-plates, all procedures performed at room temperature at a
volume of 500 .mu.L for each well. Sections were initially
permeabilized in 4% Triton X-100 (Sigma-Aldrich) in PBS for 1 hour
with subsequent PBS washing for three times. Click reaction was
performed for EdU visualization including 20 mM (+)-sodium
L-ascorbate (Sigma-Aldrich), 10 .mu.M Alexa 555-azide (Life
Technologies), and 4 mM copper sulfate (Sigma-Aldrich) in PBS.
Sections were incubated with gentle shaking for 15 minutes followed
by PBS washing. Brain sections were collected and placed on
gelatinized glass slides. All preparations were mounted with
fluorescent mounting medium (DAKO) and coverslipped.
[0101] For assessment of hippocampal neurogenesis, EdU.sup.+ cells
slides were imaged using a Leica DM5500B fluorescence microscope in
depth Z stacking was used. Cells were manually counted in the basal
layer of dentate gyrus in all 13 sections and multiplied by 6 to
obtain as estimate of the number of dividing cells per
hemisphere.
[0102] For assessment of neurogenesis in the subventricular zone
(SVZ), 13 sagittal, 50 .mu.m-thick sections were imaged using a
Leica DM5500B fluorescence microscope. In depth Z stacking was used
(images were captured as stacks separated by 4 .mu.m with a
10.times. objective). Quantity of positive cells was manually
counted in the ventral SVZ using ImageJ and the total number of
cells was normalized to the number of images taken.
Immunostaining and Telomere-Associated Foci (TAF) and
Quantifications
[0103] Paraffin sections were deparaffinized with Histoclear and
hydrated in an ethanol gradient followed by water and PBS. Antigen
was retrieved by incubation in 0.01M citrate buffer (pH 6.0) at
95.degree. C. for 10 minutes. Slides were placed in blocking buffer
(1:60 normal goat serum [S-1000, Vector Laboratories] in 0.1%
BSA/PBS) for 60 minutes at room temperature. For TAF staining,
slides were additionally blocked with Avidin/Biotin (Vector Lab,
#SP-2001) for 15 minutes each. Primary antibodies used (Table 2)
were diluted in blocking buffer and applied overnight at 4.degree.
C. The next day, slides were washed 3 times with PBS and incubated
for 30 minutes with secondary goat, anti-rabbit antibody (1:200;
Vector Laboratories #BA-1000) for TAF staining or for 60 minutes
with secondary Alexa antibody (Table 1). For TAF staining,
Fluorescein-Avidin in PBS (1:500; #A-2011, Vector Lab) was applied
to each sample for 20 minutes. Slides were washed 3 times in PBS,
which was followed by FISH for TAF detection. Briefly, tissues were
crosslinked with 4% paraformaldehyde for 20 minutes and dehydrated
in graded ethanol. Sections were denatured for 10 minutes at
80.degree. C. in hybridization buffer (70% formamide (Sigma), 25 mM
MgCl.sub.2, 0.1 M Tris (pH 7.2), and 5% blocking reagent [Roche])
containing 2.5 .mu.g ml.sup.-1 Cy-3-labelled telomere-specific
(CCCTAA) peptide nucleic acid probe (Panagene), followed by
hybridization for 2 hour at room temperature in the dark. Slides
were washed twice with 70% formamide in 2.times.SSC for 15 minutes,
followed by washes in 2.times.SSC and PBS for 10 minutes. Sections
were mounted in Vectashield, DAPI-containing mounting media and
imaged.
[0104] A single, 3 .mu.m-thick section per mouse was used for TAF
staining, while for Dcx and Plin2 staining was performed on three
10 .mu.m-thick sections 80 .mu.m-apart. To quantify periventricular
lipid accumulation 10-30 images in the periventricular region were
taken using the DM5500 widefield fluorescence microscope from Leica
with a 10.times. (for frequency of periventricular glia) or
.times.40 (for ALISE phenotype of ependymal cells) objective lens.
Number of Plin2-positive cells (for frequency of ALISE-positive
periventricular glia) or area of Plin2-positive vesicles (for ALISE
phenotype of ependymal cells) was assessed using ImageJ software.
For identity assessment of Plin2+ cells, 10 .mu.m-thick sections
were stained with combination of antibodies for Iba1 (secondary
antibody conjugated with Alexa Fluor 488), Plin2 (secondary
antibody conjugated with Alexa Fluor 594), and Vimentin (secondary
antibody conjugated with Alexa Fluor 647) and quantified for
frequency of Plin2+ astrocytes (Iba1-, Vim+) and microglia (Iba1+)
in the periventricular region. A separate staining for Plin2
(secondary antibody conjugated with Alexa Fluor 594) and NeuN
(secondary antibody conjugated with Alexa Fluor 647) was used to
determine frequency of Plin2+ neurons in periventricular region.
For TAF quantification in depth Z stacking was used (images were
captured as stacks separated by 0.4 .mu.m with .times.63 objective)
followed by ImageJ analysis.
TABLE-US-00002 TABLE 2 Antibodies used in Immunostaining Company
producing Primary Secondary Tertiary primary antibody antibody:
antibody: antibody or and catalogue origin and origin and
developing number concentration concentration system
.quadrature.-H2A.X Cell Signalling, Rabbit, 1:250 Anti-rabbit, DSC-
#9718S biotinylated, fluorescein Goat, 1:200 (Vector Lab) Perilipin
2 Synaptic Systems, Guinea pig, 1:250 Anti-guinea pig, (Plin2)
#GP46 Alexa 594 or 488, Goat, 1:1000 Iba1 Abcam, Goat, 1:250
Anti-goat, #ab5076 biotinylated, donkey, 1:1000 S100.beta. Synaptic
Systems, Chicken, 1:1000 Anti-guinea pig, #287006 Alexa 594 or 647,
Goat, 1:1000 Vimentin Abcam, Chicken, 1:1000 Anti-guinea pig,
#ab24525 Alexa 647, Goat, 1:1000 Dcx Cell Signalling Rabbit, 1:250
Anti-rabbit, (doublecortin) #4604 Alexa 594, Goat, 1:1000 NeuN/FoxO
Abcam Mouse, 1:500 Anti-mouse, ab104224 Alexa 647, Goat, 1:1000
Gfap Synaptic systems, Guinea pig, 1:500 Anti-guinea pig, #173004
Alexa 647, Goat, 1:1000 EdU Life technologies Alex555-azide,
#E10187 10 .mu.m #A20012
IHC for GFAP
[0105] Tissue distribution glial fibrillary acidic protein (GFAP)
in the brain was assessed by immunohistochemistry using an image
analysis workstation after staining with antibodies. The brain
sections were pretreated with 0.3% H.sub.2O.sub.2 methanol for 1
hour at room temperature and with normal goat serum for 1 hour at
room temperature. Each specimen was incubated with the primary
antibody overnight at 4.degree. C. The primary antibody used in
this study and the dilutions were as follows: rabbit anti-cow glial
fibrillary acidic protein (GFAP) [1:800, DAKO, Denmark].
Immunohistochemistry was performed using the VECTASTAIN ABC System
(Vector Laboratories Inc., Burlingame, Calif.) with the
avidin/biotin peroxidase complex (ABC) method. Negative controls
included replacement of the primary antibodies with normal rabbit
serum [1:200, DAKO]. The immunoreactivity to rat positive control
specimen of the primary antibodies was determined before use.
RNA In Situ Hybridization
[0106] RNA-ISH was performed after RNAscope protocol from Advanced
Cell Diagnostics Inc. (ACD). Paraffin sections were deparaffinized
with Histoclear, rehydrated in graded ethanol (EtOH) and
H.sub.2O.sub.2 was applied for 10 minutes at RT followed by two
washes in H.sub.2O. Sections were placed in hot retrieval reagent
and heated for 15 minutes. After washes in H.sub.2O and 100% EtOH
sections were air dried. Sections were treated with protease plus
for 30 minutes at 40.degree. C., washed with H.sub.2O and incubated
with target probe (p16) for 2 hours at 40.degree. C. Afterwards,
slides were washed with H.sub.2O followed by incubation with AMP1
(30 minutes at 40.degree. C.) and next washed with wash buffer (WB)
and AMP2 (15 minutes at 40.degree. C.), WB and AMP3 (30 minutes at
40.degree. C.), WB and AMP4 (15 minutes at 40.degree. C.), WB and
AMP5 (30 min at RT) and WB, and, finally, AMP6 (15 minutes at RT).
RNAscope 2.5 HD Reagent kit-RED was used for chromogenic labelling.
After counterstaining with haematoxylin, sections were mounted.
[0107] For analysis of cytokines (Il-6 and Cxcl1) sections were
co-stained with antibodies for Plin2 and S100.beta.. Briefly,
following chromogenic labelling for cytokines, sections were washed
3 times in TBS for 5 minutes each followed by blocking in 0.1% BSA
in PBS for 30 minutes at RT. Sections were incubated overnight with
primary antibodies at 4.degree. C. Next, sections were washes 3
times in TBS for 5 minutes each followed by secondary antibody
incubation for 1 hour at RT. After 3 TBS washes sections were
mounted using ProLong Gold mounting media containing DAPI. Probes
used: Cdkn2a: 411011, Il-6: 315891, Cxcl1:407721 (all from
ADC).
[0108] For all RNA-ISH experiments data was analyzed by quantifying
the % of positive cells (which means each cell containing at least
1 focus was counted as positive).
Statistical Analysis
[0109] Data are presented as mean.+-.SEM for all data. All
statistical analyses including testing the normality of data
distribution were performed using GraphPad Prism 7.01 and a P value
<0.05 was considered as significant. The study was designed to
compare change in parameters between lean and obese animals and
between obese and obese treated animals. All data were assessed for
normality using D'Agostino & Pearson normality test (for
n>7) or Shapiro-Wilk normality test (for n 7.gtoreq.n>3). For
2-group comparisons and planned comparison 2-group comparisons
(were used where appropriate when main effects were significant
without significant interactions) data was further tested for
equality of variances using F test. For non-normally distributed
datasets (p<0.05 in D'Agostino & Pearson or Shapiro-Wilk
normality tests) Mann-Whitney U test was used. For normally
distributed datasets Welch's t-test (if p<0.05 in F test) or
Student's t-test was used. For 2> groups comparisons one-way
ANOVA with Tukey's multiple comparison test was used. For datasets
split on two independent factors two-way ANOVA was used.
Correlations were assessed using Pearson's (for datasets of normal
distribution) or Spearman's (for datasets of non-normal
distribution) rank correlation test.
OTHER EMBODIMENTS
[0110] It is to be understood that while the invention has been
described in conjunction with the detailed description thereof, the
foregoing description is intended to illustrate and not limit the
scope of the invention, which is defined by the scope of the
appended claims. Other aspects, advantages, and modifications are
within the scope of the following claims.
* * * * *